In 2005, the first next-generation sequencer (NGS) was introduced to the biomedical world and ushered in an exciting era for medicine. Irrespective of disease, these technologies hold the promise of personalized medicine through the stratification of patients based on their genomes. As the pharmaceutical industry moves away from producing blockbuster drugs and toward developing targeted therapeutics that appreciate genomic and molecular variations among patients, the need for a diagnostic tool that enables cost-effective, rapid and accurate sequencing has become ever more pertinent.
During the 1970s, two competing teams, led by Fredrick Sanger and Walter Gilbert, published seminal articles detailing the first approaches to DNA sequencing. Gilbert's technique allowed chemical modification and cleavage of radioactively end labeled DNA fragments that were subsequently separated by gel electrophoresis and visualized on film autoradiography. Sanger's method used a chain termination technique where dideoxynucleotides caused base specific termination of DNA sequences. By replacing radioactive primers with fluorescent ones and incorporating capillary electrophoresis over slab gel, Sanger's methodology went on to become the gold standard in both research and clinical laboratories . It was this highly accurate and automated technique on which the 13 year long, $2.7 billion Human Genome Project (HGP) was completed. However, as a solution for analyzing complex diploid genomes, Sanger's lack of scalability means the platform falls short on throughput and cost, prompting the development of NGS technologies.
Currently, the NGS market is dominated by three players: Life Technologies, Illumina and Roche. The main difference between these technologies and Sanger is the ability to conduct massively parallel sequencing, allowing for the precipitous decline in cost and time to sequence with the resultant generation of billions of sequence reads. Each of these NGS instruments employs a unique proprietary chemistry based on a similar concept - identifying signals emitted, either by light or H ion fluxes, during the incorporation of bases into the DNA template by polymerase. The original NGS platforms (SOLiD, HiSeq, GS FLX) are primarily research focused machines producing huge amounts of sequence data in a matter of days. With the need for even quicker turnaround times for the clinic, and smaller instrument sizes in the labs, the newer versions such as the Ion Torrent's PGM from Life Technologies, Illumina's MiSeq and Roche's GS Junior have allowed NGS to become part of the clinical diagnostic arena.
The Clinical Setting
The application of NGS in the clinical setting is already becoming a reality, particularly in the field of cancer molecular diagnostics, driven by the need to stratify patients based on their genomic profile. The development of targeted therapies, such as Tarceva for EGFR mutation positive lung adenocarcinoma, precipitated the adoption of single gene tests using immunohistochemistry, DNA microarray or RT-PCR. As our understanding of the cancer genome increases, an abundance of new targets and clinical trials for therapeutics have sprung up, expanding the treatment armamentarium for oncologists. Given the need to interrogate a larger portion of the cancer genome and identify a variety of aberrations, including translocations, NGS-based applications have been developed providing prognostic and predictive information to support decisions around treatment and the selection of therapies.
Another area where NGS technology will impact clinical care is in the screening of newborns for Mendelian disorders. Physicians typically rely on phenotype -genotype correlations, using single gene tests to confirm their diagnoses. However, Mendelian conditions can present in heterogeneous ways with the same phenotype being caused by multiple genes, including ones yet to be described. Here the application of NGS can have a tremendous impact on the diagnosis and speedy management of these life-threatening conditions. The recent successes of Children's Mercy Hospital in identifying 592 rare childhood diseases in about 50 hours provides an example of how this technology is progressing in the clinical research environment.1
In the area of prenatal diagnosis, innovative testing modalities based on NGS may change traditional practice. The Sequenom Center for Molecular Medicine currently offers MaterniT21, a non invasive prenatal test for trisomy 21 that detects small increases in fetal DNA material for chromosome 21 in the maternal blood plasma. This non-invasive test (and others like it for trisomies 13 and 18) is highly sensitive and specific and will find an appropriate place in the management of pregnancies.2
NGS technologies are also being applied in the field of infectious disease and public health surveillance. Recently, the National Institutes of Health published a study where real time sequencing was used during an outbreak to track transmission. During the 2011 outbreak of the fatal hemorrhagic intestinal infection that swept across central Europe, an Ion PGM from Life Technologies was used to quickly identify the deadly strain of E. coli. In the hospital setting, NGS technology could prove advantageous for tracking the spread of MRSA, and efforts are under way in the UK to catalogue all the MRSA strains using NGS into a national database.
Adoption of NGS
As with any diagnostic poised to enter the clinical space, the widespread adoption of NGS will continue to be slow unless significant steps are taken to address the ethical, technical, interpretive and regulatory issues that surround the technology. The most pertinent of these is the ability to analyze the gigabases of data generated analysis, which can be very costly. This requires the expertise of a new breed of professional, the clinical bioinformatist, as well as physicians trained in such data analysis to distill the most relevant and clinically actionable information from this voluminous data.
In addition, assurances and control around quality are vital to ensure that NGS platforms do what they say on the box. In this vein, multiple groups, including the Centers for Disease Control Division for Laboratory Science and Standards as well as the College of American Pathologists, have been working on compiling a checklist of requirements and quality metrics for the precision, accuracy, sensitivity, specificity and reportable ranges of NGS technologies. Such initiatives will ensure that reimbursement issues surrounding this technology are also addressed.
Dr. Billings is chief medical officer and Dr. Shather is a medical associate with Life Technologies.
Saunders CJ, et al. Rapid whole-genome sequencing for genetic disease diagnosis in neonatal intensive care units. Sci Transl Med 2012;4(154ra135).