Next Generation Sequencing and Personalized Genomic Patient Care

I’m sure that everyone has heard about next generation sequencing (NGS). But why exactly is it a big deal? Even though I have spent a significant amount of time at the bench performing “wet lab” basic science research and was acquainted with the term, I did not have practical hands-on experience with NGS prior to residency. It was not a readily accessible technology during my biomedical research days prior to medical school and so I did not entirely grasp the full power of this then disruptive technology until I was a pathology resident, and even more so, as an applicant for molecular genetic pathology (MGP) fellowships.

All of us should have previously learned about the “gold standard,” Sanger sequencing. This method combined irreversible dideoxynucleotide chain termination with a detection method such as gel electrophoresis, or on a larger, automated level, capillary electrophoresis. It was powerful because it allowed us to read the genomic map that directs cellular life, albeit only one sequence at a time. It served as the mainstay for more than a quarter of a century and still is employed for smaller scale sequencing or for long (>500 bp) stretches of DNA.

In the 1990’s, initial methods of massively parallel signature sequencing (MPSS) and pyrosequencing began to appear which would lay the groundwork for today’s massively parallel sequencing (MPS), also known as second generation sequencing, or more popularly as NGS. The two most commonly utilized NGS platforms to date are based on semi-conductor technology for the Ion Torrent (now Life Technologies) and reversible dye-terminator, sequencing based synthesis (SBS) technology for the Illumina platforms.

The power of NGS comes from its ability to simultaneously sequence 1 million to 43 billion short reads (400-500 bp each). The Human Genome Project took over a decade to complete and cost nearly $3 billion whereas NGS can sequence the same genome for a cost on the order of $1000’s, a cost that is further decreasing as the technology is refined. When I was working in research (eons ago), we had nitrocellulose based dot arrays where each “dot” represented multiple copies of a specific cDNA sequence and which helped us to build expression profiles for our particular area of study. This would be analogous to analog technology and NGS would now be considered a digital version.

With conventional PCR, we could only amplify one target sequence per sample reaction. The results were also only qualitative. The results only measurable after multiple cycles of denaturation, annealing, and extension as amplified product of the expected size or no amplified product. Then came along real-time or quantitative PCR (qPCR) which some people refer to also as RT-PCR (although this is a confusing term for people like me who were around before qPCR and think of RT-PCR as meaning reverse transcription PCR). The power of qPCR was that within the linear range of detection, we could now quantitate the amount of product present in real time. NGS also provides the resolution of qPCR in terms of quantitation.

So, as a technology, NGS nicely combines and refines some (but not all) characteristics of multiple technologies with large scale profiling. But for a non-molecular person, what is the relevance? Obviously, it has taken us some time to get to this point, even though the Human Genome Project was completed in 2003 (it started in 1990). We needed time for biomedical and translational research to provide us with clinical significance to the mutations and genetic aberrations NGS could identify. We also needed to develop tools to distinguish true mutations with clinical significance from benign polymorphisms present in our population and to build our bioinformatics support.

But here we are, stepping into the new frontier of personalized genomic medicine. Sure, there is a lot of hype surrounding it and these promises will take time to keep. But these are exciting times for someone like me who fell in love with the molecular dance that plays out within our cells. One of the reasons that I want to complete an MGP fellowship is to get more hands-on practical knowledge of the nuances of NGS from a technical standpoint but also to collaborate with other physicians in directing patients to the correct clinical trials and targeted treatment – and therein, is where the power of NGS really lies. For someone who may never get to meet the patient, at least for me, there’s probably no greater satisfaction than knowing you had a pivotal part in helping a patient more effectively combat their disease. Personalized genomic medicine is another step in that direction.

[12/12/2014: edited to fix a few inaccuracies. We apologize for the error.]


-Betty Chung, DO, MPH, MA is a third year resident physician at Rutgers – Robert Wood Johnson University Hospital in New Brunswick, NJ.

Enterovirus D68

Over the past few weeks, hospitals around the country have seen a sharp uptick in cases of respiratory distress in children. The majority of patients test positive for Enterovirus D68, and most seem to have a history of asthma.

Only select laboratories test for this strain of enterovirus. If a suspected case comes to your facility, contact the CDC or your local health department for information about specimen collection and shipping.



Viral DNA Testing, Cervical Cancer, and Cytology

An FDA panel recently recommended use of Roche’s Cobas HPV DNA test as the initial screening tool for cervical cancer instead of the ubiquitous Pap smear. The panel found that “A negative HPV result predicted a lower 3-year risk of ≥CIN3 than did a negative cytology result, suggesting that using HPV as the primary test is superior to cytology for cervical cancer screening.The low 3-year CIR for a negative HPV result also confirmed the safety of a 3-year interval for HPV primary screening and officer clinicians more confiedence in a negative HPV result than a negative cytology result.”

While this isn’t a final FDA guideline, it’s conceivable that clinicians could alter their practice based on these findings. Of course, this will affect the cytology and molecular diagnostics departments, as well. What do you think? Is HPV viral testing the way to go? Or should we stick with the test that’s been around for decades?

ASCP Call for Abstracts

Do you want to present your research at a national meeting? The American Society for Clinical Pathology is currently accepting abstract submissions for their Annual Meeting. This year it’s in October in Tampa, Florida. Soak up the sun while presenting your work and networking with your peers.

Are Pathologists and Primary Care Physicians Ready for the Genomic Era and Personalized Medicine?

I was reading about the FDA’s recent crackdown on 23andme to stop marketing their saliva based whole genome testing and interpretation service. Rather than resist, 23andme decided to comply and is currently in “talks” with the FDA so that they can complete the process for FDA validation and again begin to market their kits and testing. For now, they can continue to provide their genealogy testing and whole genome sequencing without interpretations.

Currently, in some academic research centers, whole genome or exome sequencing via next generation sequencing (NGS) methods is utilized on a limited basis by researchers and clinicians to identify pathogenic mutations. NGS and bioinformatic analysis methods continue to steadily improve and costs have been decreasing. However, there are limitations and barriers to widespread use at this point. These include but are not limited to: 1) widely used databases such as the Human Gene Mutation Database (HGMD) and the Online Mendelian Inheritance in Man (OMIM) still only contain information that only covers a fraction of the human genome, 2) more research is still needed to identify more variants mutation-disease associations, and 3) most mutations identified fall under the category of “unknown clinical significance”.

Tools such as NGS, despite its improvement over previous technology, still cannot identify large deletions or copy number variations (CNV) and is a technology not accessible, cost-wise and support-wise, to most health care institutions. Despite all of this, primary care physicians, even now, still may be confronted with patients who bring them their genomic screening results, whether obtained from commercial services provided by companies like 23andme or through molecular testing through a health care institution. But today’s physicians, including primary care physicians and pathologists, were not trained in medical school to understand how this testing is performed or the significance of these results. But the time is coming, and maybe sooner than we realize, when we will have to deal with such testing on a daily basis.

So, it is imperative that we train our doctors and doctors-in-training now to be ready for when that time comes. But, my question this week is “How should we go about it?” Additionally, who should compose the health care team to provide guidance and counseling to patients once results are available? And who should regulate how testing should be done and what information should be included in results reporting? Leave me a comment if you have an opinion or any ideas.


Betty Chung, DO, MPH, MA is a second year resident physician at the University of Illinois Hospital and Health Sciences System in Chicago, IL.

Personal Genome Testing and You

The recent events with 23andme and the Food and Drug Administration have brought personal genomic testing into the spotlight. In case you haven’t been following the case, the FDA wrote a warning letter to 23andme—a company that will access various points on your genome and give you the results for around $99—that basically states that because the tests diagnose, mitigate, or prevent disease they require regulatory clearance. The FDA also said false positive results for certain breast or ovarian cancer markers could lead to unnecessary preventative surgery.

Since receiving the letter, 23andme as stopped marketing their genetic testing service. At this writing I’m not aware of the status of ongoing testing or if the company is still accepting new samples. 23andme has 15 working days from the date of the letter—which would be 12/13—to let the FDA know how they’re working to resolve the noted issues.

How does this affect laboratory medicine? On the face of it, not that much. Yes, clinical laboratory scientists and pathologists could lose their jobs of the 23andme labs were forced to close their doors. The field of personal genome testing is a relatively new one—23andme began testing in 2008—but even so, it’s important to realize that this type of testing can positively affect laboratory professionals and pathologists.  More laboratories equal more jobs, after all, and not just for bench techs and pathologists, but for consultants, inspectors, and administrators as well. I know that I’ll be watching to see how this plays out.

-Kelly Swails