During the past few years, next generation sequencing (NGS) has seen widespread growth into expanded applications and experienced increased media attention, taking sequencing from cutting-edge research to dinner table conversation. The exponential growth in market breadth has paralleled advancements in technology, particularly in the areas of benchtop sequencing and high-throughput systems, bringing increased automation, ease of use, and decreased cost. This trend is facilitating access to sequencing for laboratories of all sizes, enabling leaps in genetic research across varied fields of biology, from agriculture to energy to human disease research.
Perhaps most poised to benefit from the latest advances in sequencing technology are applications in translational research, the process of translating scientific discoveries into routine applications, including clinical diagnostics. Genetic profiling to determine more effective and more personalized treatments for disease, such as companion drugs and diagnostics, is becoming the new reality. Today, top researchers and medical centers are taking NGS data toward a future of genomically informed medicine. The ability to examine consequential portions of an individual’s genome, or the genomes of the bacteria and viruses in and on them, in order to identify individualized risk predictions and treatment decisions, is finally within reach and has the potential to impact clinical decision making globally.
Unraveling tumor genomes
Next generation sequencing is playing a transformational role in cancer discovery research. Providing new insights into pathologic signaling pathways is one example of advancements made through NGS which were previously impractical due to technological limitations.1 Compared to traditional Sanger sequencing approaches, targeted massively parallel pyrosequencing provides greater sensitivity in detecting low-level genetic variants and also allows unambiguous haplotyping due to the clonal nature of the reads. These advantages are particularly relevant for tumor genetics, where the ability to tease apart the constituent DNAs in a mixed population of tumor cells is especially useful in identifying genetic variants in complex and heterogeneous diseases.
Moving forward, targeted next generation sequencing holds tremendous potential for rapid, comprehensive mutational profiling to elucidate complex signatures and tumor variation. Targeted sequencing substantially improves the cost-effectiveness of sequencing by focusing the available sequencing bandwidth on the portions of the genome that are most likely affected. At Thomas Jefferson University, a team led by Dr. Stephen Peiper has explored the use of targeted sequencing with a long read benchtop NGS platform, combined with sequence capture technology, to analyze a panel of genes associated with colorectal cancer. By sequencing the genes in question thousands of times in parallel in a single experiment, the team was able to compare tumor samples with paired normal samples to identify acquired somatic genetic variants. “As an initial transition from Sanger sequencing to next generation sequencing, it has been comforting to look at long reads,” explains Dr. Peiper. “It has given us more of a sense of what we were seeing rather than having a black box and getting bioinformatics back.”
In the current age of next generation sequencing, the largest hurdle facing translational research laboratories is not generating the sequence data, but rather interpreting it. While next generation sequencing can efficiently generate piles of data, additional innovation is needed to improve researchers’ ability to make sense of this data so the technology is feasible for routine use.
Deconstructing the human microbiome
Another area where next generation sequencing is playing a significant role is human microbiome research. Using NGS technology, it is now possible to comprehensively characterize the diverse microbial communities that inhabit the human gut, mouth, and skin and then compare these communities in diseased and healthy individuals. The latest studies are forcing clinical researchers to more carefully consider the role that these human bacterial communities play in almost all aspects of human health, development, and disease.
One of the largest efforts in this area is the Human Microbiome Project, a National Institutes of Health-led consortium comprising more than 200 researchers and 80 research institutes. With the end goal of better understanding the effects of disease and other changes on human microbial communities, the researchers used primarily targeted sequencing of 16S rRNA (a common bacterial signature) to study 300 healthy volunteers and evaluate microbial diversity across various body sites over time. The result is the largest collection to date of the microbial communities in healthy individuals.2
While further away from routine medical practice, microbiome studies still hold tremendous medical potential. For example, in the future, doctors may rely on more discriminating methods of treating infections based on their microbial composition, rather than broad antibiotics.
Infectious diseases and viral diversity
Closer to the clinical world, next generation sequencing also enables more accurate resolution of the diversity of viral communities that are infecting an individual, much like the resolution of mixed populations of cells in cancer. Viruses with a high mutational rate, such as Human Immunodeficiency Virus (HIV), are characterized by diverse sub-strains, many of which can be present in an infected individual at any given time. Next generation sequencing has shown great potential for illuminating the evolutionary pressures that occur during the treatment of an infection and showing how they ultimately can lead to the emergence of drug-resistant viral strains.3 By using this sequencing information, clinicians may in the future be able to detect drug resistance far earlier, enabling the timely adoption of therapeutic interventions that are tailored to the specific infection present within an individual.
Remaining challenges
Despite the tremendous progress in translational research, several significant challenges must be overcome before next generation sequencing technology can become a part of routine clinical practice. Manufacturers, laboratorians, and regulatory agencies first need to reach greater consensus in areas such as the criteria for selection and quality of samples, data quality and reproducibility, workflow standardization, bioinformatics handling, regulatory guidance, and the clinical significance of variants. In the meantime, we can expect research using NGS technology to drive rapidly toward a future that holds greatly enhanced potential for personalized healthcare.
References
- Kan Z, et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature. 2010;466(7308):869-873.
- Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature. 2012;486(7402):207-214.
- Simen B, et al. Low-abundance drug-resistant viral variants in chronically HIV infected, antiretroviral treatment-naive patients significantly impact treatment outcomes. J Infect Dis. 2009;199(5):693-701.
Benjamin Boese, PhD, is International Product Manager at 454 Life Sciences, a Roche company that develops and commercializes the GS Junior and GS FLX Systems for highthroughput DNA sequencing.
