March 1, 2014 (Vol. 34, No. 5)

Next-gen sequencing (NGS), the media-friendly moniker for automated massively parallel DNA or RNA sequencing technology, has made the leap from a powerhouse tool for molecular biology research and drug discovery and development to the clinical arena.

Not an easy feat for a complex, expensive technology that generates huge amounts of data requiring intensive analysis and interpretation.

The first next-gen sequencer to receive FDA clearance is the Illumina MiSeqDx system, with other NGS instruments hot on its heels. NGS-based diagnostic tests and clinical research tools are already beginning to transform fields such as prenatal testing and oncology, even as predictions for the scope of their applicability continue to broaden.

In a Perspective piece published December 19, 2013 in the New England Journal of Medicine, Francis Collins, M.D., Ph.D., and Margaret Hamburg, M.D., wrote about the first FDA authorization of a NGS instrument: “Clinicians can selectively look for an almost unlimited number of genetic changes that may be of medical significance. Access to these data opens the door for the transformation of research, clinical care, and patient engagement.”

Illumina designed the MiSeqDx system specifically for clinical laboratories, making it affordable and easy to use to broaden its applicability.

“At about the $10,000 threshold we started to see early clinical adoption of next-gen sequencing,” says Greg Heath, Ph.D., svp of in vitro diagnostics at Illumina. Initial applications in rare inherited diseases in children and end-stage cancer were driven primarily by medical need, and technological advances expanded the use of NGS into the area of noninvasive prenatal testing for aneuploidies, bringing with it a great deal of support from the medical community and healthcare reimbursers, according to Dr. Heath.

“I think NGS will displace a lot of PCR-based tools,” predicts Dr. Heath.

The most prominent emerging areas for clinical NGS growth, in his view, include genetic disease, with a particular emphasis on reproductive genetics, and oncology, in which “certain unique performance characteristics of the technology will make it possible to solve some of the fundamental problems in cancer,” such as the heterogeneity of tumors and the difficulty of working with FFPE samples.

Other clinical applications where NGS may have a near-term impact are in transfusion and transplantation medicine. A bit farther off, applications will likely emerge related to methylation in cancer and immunosequencing.

Emerging Standards

Nazneen Aziz, Ph.D., director of the College of American Pathologists (CAP), is an expert in genomics who has had a first-hand view of molecular genetics research since before the Human Genome Project, both in Harvard Medical School’s faculty and in the biotech industry, focusing on the discovery of new genes abnormally regulated in inherited disease, as well as on human genetic markers for diabetes and oncology drug development and for genetic tests to understand the risks for common diseases such as osteoarthritis and osteoporosis.

Since joining CAP, Dr. Aziz formed a new committee on NGS, which has “come up with the first global standards on next-generation sequencing for accrediting labs offering this technology as a clinical test.” CAP released its initial standards in July 2012, with a revision in 2013, and will distribute another revision in 2014. The standards outline requirements for documentation requirements, validation for the wet bench and bioinformatics analysis, data storage, quality management, and other important considerations for the clinical workflow.

NGS is a term used to describe high-throughput, massively parallel sequencing, in contrast to the lower throughput, earlier method commonly known as Sanger sequencing. As Dr. Aziz explains, the overall cost of NGS tests is about the same as for Sanger sequencing, but NGS yields much more sequence information in a single run, making it more efficient with a much lower per base cost.

A number of companies offer NGS platforms and instruments for research use, among them Illumina’s HiSeq and MiSeq systems, Life Technologies’ Ion Torrent platform, Pacific Biosciences’ SMRT Sequencing-based PACBIO RS II instrument, and Complete Genomics’ nanoball array-based technology. (Life Technologies’ acquisition by Thermo Fisher Scientific was finalized February 4, 2014; Complete Genomics is a wholly owned subsidiary of BGI-Shenzhen.)

The Illumina MiSeq Dx platform is the only one as yet granted FDA marketing authorization for clinical use. While the technology and chemistries in these platforms and others are quite different, “the overall processes are similar” in the sense that a clinical lab would use them essentially the same way—loading samples and generating sequence data—says Dr. Aziz. The bioinformatics pipelines of the available NGS platforms differ in some key aspects, she notes, but the information output is the same—sequence data.

Early Application

Providing an overview of the earliest clinical applications of NGS, Dr. Aziz highlights the identification of variants in rare inherited conditions in children. Instead of putting a child with a constellation of nonspecific symptoms and no clear diagnosis through a barrage of tests, sequencing either the whole genome or the exome can help clinicians determine the genetic cause of the child’s condition in about 20–25% of cases. In some cases, they will discover a new mutation or gene variant and be able to characterize a novel monogeneic disorder.

Another early application of NGS is cancer genomic analysis. “Everyone’s cancer is different at the molecular level,” says Dr. Aziz. “Cancer is no longer a disease of the tissue—like breast cancer or pancreatic cancer; you need to look at the molecular profile of each tumor.”

NGS can quickly reveal the somatic variants in cancer. Resequencing throughout the course of treating a patient with cancer can identify new mutations that may be responsible for the development of drug resistance or guide treatment decisions and selection of new and experimental compounds when conventional therapies fail.

In the future, NGS applications in the area of infectious diseases will likely increase, suggests Dr. Aziz. The technology offers advantages for identifying strains of microoganisms causing outbreaks, for example, or in personalizing therapy by testing a strain for resistance to antibiotic or antiviral medications.

Prenatal Testing

Dirk van den Boom, Ph.D., evp of R&D and CSO, describes Sequenom Laboratories’ MaterniT21™ PLUS noninvasive diagnostic test for fetal chromosomal abnormalities as “the first real example of applying next-generation sequencing in a clinical setting in a high-throughput fashion.” More than 148,000 tests were performed in high-risk pregnant women in 2013, avoiding the risks associated with amniocentesis or chorionic villus sampling to access fetal DNA. The test sequences cell-free DNA in a sample of maternal blood—which includes fragments of fetal DNA—matches the fragments to the chromosome from which they derived, and essentially determines if a chromosome is present in greater than the normal diploid amount.

“NGS is a good tool for this because of the number of data points you can generate,” says Dr. van den Boom. Sequenom Laboratories implemented the test on Illumina’s HiSeq platform and initially reported on chromosome 21.

Prenatal testing is a big market, with an estimated 750,000 high-risk pregnancies each year in the U.S. alone. “We started with trisomy 21, but we chose a whole-genome approach so we could add content as we have the clinical data and see the medical need, “ he adds.

Sequenom next added the capability to detect trisomy 18 and 13 and sex chromosome aneuploidies, and toward the end of last year introduced the Enhanced Sequencing Series as an additional feature to the MaterniT21 PLUS test, which added trisomy 16 and 22 as well as larger clinically relevant chromosomal microdeletions such as those associated with DiGeorge syndrome, Cri-du-chat syndrome, and Prader-Willi/Angelman syndrome.

The ability to generate a full fetal karyotype by using NGS to analyze a maternal blood sample “is fundamentally in reach within the next couple of years,” predicts Dr. van den Boom. As the company continues to expand its prenatal testing portfolio, it has also begun to explore the power of applying NGS to DNA fragments in the bloodstream to the detection of cancer.


Sequenom Laboratories’ MaterniT21 PLUS test is performed at one of the company’s U.S.-based facilities—a lab in San Diego, CA or a lab in Morrisville, NC (near Raleigh). The turnaround for results is about seven days from receipt of the sample in the lab.

Oncology Testing

According to Andy Felton, director of Life Technologies’ genetic systems division, “oncology testing will be a huge potential market” for NGS. Unlike PCR and Sanger sequencing, it gives you the ability to look at many mutations in parallel—single nucleotide changes, copy number variations, and gene fusions, for example.

Life Technologies’ Ion PGM System is currently a research use only instrument; the PGM DX platform in development will target the clinical setting and is in the process of completing testing for future registration and listing with the FDA. The company continues to streamline and enhance the automation and ease of use of its research platform, advances it intends to apply to a future clinical testing system.

Citing the speed of its NGS technology—with a turnaround time of a few hours—and the scalable output as key advantages of the Ion Proton System, Dr. Felton also highlights the ability of the platform’s AmpliSeq technology to accommodate samples as small as 10 ng, as may be common in future clinical testing of FFPE tissue samples.

From a research perspective, the goal of NGS is to obtain as much sequence data as possible—to find that SNP, translocation, or other variant that correlates with disease, but “we ask the opposite question as the researcher,” says Gitte Pedersen, CEO of Genomic Expression. To use RNA-seq in the clinical setting, for example, we want to know “what is the minimum amount of information you need to extract from your sample in order to identify and quantify all of the RNAs.” RNA-seq by itself is not a clinical application, because depending on how the test is analyzed, and who analyzes it, the results can be different, asserts Pedersen.

To reduce the time, cost, data burden, and complexity of RNA-seq applications to make them faster, easier, and more amenable for use in the development of a companion diagnostic for instance, Genomic Expression produced kits and accompanying software for performing automated RNA-seq that are platform-agnostic and work on existing and emerging NGS instruments. Based on a bait-free target filtering sample preparation method, the process from sample prep to results takes less than a week and generates a data file of 30 MB that can be attached to an electronic medical record.

Pedersen describes how pharma companies can use RNA-seq to develop companion diagnostics for stratifying patient populations for clinical testing of oncology drugs, for example. As data is collected it can be used to perfect the evolving algorithms and “optimize the definition of the target population based on data points.”

This exercise to link a companion diagnostic to a cancer therapeutic may have required an array of different markers and multiple testing platforms, but RNA-seq can turn that complexity “into a math exercise with a very high probability of success on one platform with one assay,” Pedersen adds.

“With the approval of the Illumina MiSeq for clinical applications, we are 90% there, the platforms are there; the last 10% is to develop the algorithms. Our business model is to partner around the last 10%—the content, leveraging our access to fully annotated clinical samples.”

Pedersen emphasizes the scalability of the Genomic Expression technology, and its broad range of potential applications. How sample- or disease-specific it will be remains to be seen. “We know it works in breast cancer,” she says, and have demonstrated that it could also work in a number of other cancers. Other promising application areas are organ transplantation rejection, cardiovascular and central nervous system disorders, and autoimmune diseases.


Genomic Expression asserts that its bait-free RNA sequencing process—which encompasses sample preparation, sequencing, and analysis—can produce results in less than a week while generating a data file of just 30 MB that can be attached to an electronic medical record.

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