September 1, 2014 (Vol. 34, No. 15)

Neil McKenna, Ph.D.

Recent years have seen rapid advances in the capacity of molecular biological techniques to simultaneously interrogate multiple targets on omics platforms.

While these techniques are well established in basic research, they have more recently gained a foothold in clinical diagnostics, with an increasing number of laboratory tests incorporating some type of high-throughput or global-scale molecular analysis. Of these techniques, the one that arguably has the greatest potential to revolutionize our approach to diagnosing disease and tailoring therapies is next-generation sequencing (NGS).

NGS, which refers to a constellation of techniques in which DNA or RNA fragments are sequenced in parallel, offers significant increases in speed, scalability, and resolution over traditional sequencing methodologies. Testament to the considerable interest in NGS is the increasing number of conferences exclusively devoted to the topic.

One such event, GTCBio’s “Next-Generation Sequencing” conference, was recently held in San Diego. Presenters showcased the application of NGS-based diagnostic platforms in a variety of clinical settings.


Preimplantation Genetic Screening

Varied genetic prenatal conditions impact pregnancy and fetal development. Accordingly, preimplantation genetic screening (PGS) has emerged as an important clinical tool for identifying chromosomal aberrations.

Traditional prenatal screens have a number of limitations. Some screens, such as amniocentesis or chorionic villi sampling, are invasive; others, such as ultrasound or biochemical screening, are less invasive but limited in their sensitivity and specificity.

“In contrast to these techniques, noninvasive PGS-based on NGS has both superior detection sensitivity and specificity for chromosomal abnormalities,” said Keith Jones, Ph.D., vp of development at Illumina. “The Illumina verifi® test detects greater than 99% of all true-positive cases and has a cumulative false-positive rate of <0.2%.”

The verifi test uses sequence information from across the genome. This approach, Dr. Jones suggested, allows for the rapid adoption of additional tests that may find abnormalities not readily detected using traditional screening approaches. Such abnormalities include sex chromosome aneuploidy, microdeletions, trisomy 9, and trisomy 16.

Approximately 1.3 million in vitro fertilization (IVF) procedures are performed globally each year; however, only 25% of the procedures meet with success. The low success rate is usually attributed to complicating factors associated with advanced maternal age and chromosomal aneuploidy in the embryo.

“The aim of PGS in the IVF setting is to select chromosomally balanced embryos during the IVF process and ensure that only euploid embryos—those with a normal number of chromosomes—are implanted during IVF procedures,” explained Dr. Jones. He added that PGS has been shown to improve implantation success rates and reduce the number of high-risk pregnancies associated with multiple egg transfers.

In Illumina’s VeriSeq™ PGS platform, genomic DNA from a single cell is amplified and sequenced to provide a genome-wide view of the copy number state of the embryo. The protocol takes less than a day and allows multiplexing of up to 24 samples per sequencing run, translating to an increased likelihood of identifying a viable embryo and decreasing the time between biopsy and an answer. “The broad dynamic range derived from the sequencing data makes interpretation clear with a high degree of confidence,” Dr. Jones asserted.


Illumina scientists monitor a high-throughput sequencing run with the company’s HiSeq 2000, which provides real-time progress indicators.

Testing for Minimal Residual Disease

“The overarching theme in the NGS molecular diagnostics space is that robust clinical validation is a must,” said Martin Moorhead, Ph.D., vp of computational biology and software development at Sequenta. The company’s LymphoSIGHT platform is an NGS-based immune repertoire analytical solution that combines multiplex PCR assays and informatics algorithms to interrogate rearranged immunoglobulin and T cell receptor genes.

“Our PCR process targets the CDR3 region and the immediate surrounding sequence, yielding amplicons that are typically around 150 base pairs in length, which is ideal for NGS analysis,” Dr. Moorhead pointed out. In the assay, sequencing of rearranged B or T cell receptor gene amplicons from patient lymphocyte samples allows for an absolute quantification of the number of each clonotype—cells all sharing the same rearranged receptor sequence in the original sample.

“The first clinical product we developed using the LymphoSIGHT platform is the ClonoSIGHT test for measuring minimal residual disease (MRD) in patients with blood cancers, including diffuse large B cell lymphoma, multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and mantle cell lymphoma,” stated Dr. Moorhead. MRD refers to cancer cells that may remain in the body of a person with lymphoid cancer after treatment, and is the leading cause of relapse in this condition.

Testing for MRD can help determine whether treatment has been successful, provide important information about patient prognosis, and help guide additional treatment decisions. At its essence, the ClonoSIGHT assay compares cancer cell DNA sequences generated using the LymphoSIGHT platform in a diagnostic sample with those in follow-up samples to determine the presence of residual cancer cells.

“ClonoSIGHT test results, which are generated in seven days using our CLIA-certified, CAP-accredited laboratory, are provided in a simple, actionable report,” added Dr. Moorhead. “[The report] shows a patient’s MRD status and level as well as MRD trends over time.”


Interrogating the Microbiome

“Because our bodies are not sterile, and there are microbes and microbial communities throughout our anatomy, there is the possibility that the microbiome has a role in many diseases,” said George Weinstock, Ph.D., leader of the Human Microbiome Project and professor at the Jackson Laboratory. The human microbiome refers to the aggregate of bacteria, fungi, and archaea on the skin, in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts.

“A causative role for the microbiome has been established in only a few diseases, such as dental caries,” Dr. Weinstock continued. “But changes in the microbiome have been correlated with a large group of disease conditions.”

Drawing connections between these diseases and specific microorganisms has been difficult because most human-associated microbial species have never been successfully isolated in the laboratory. Such connections, however, may emerge from microbiome research bolstered by NGS.

“The ability to sequence all the nucleic acids extracted from a clinical sample and then analyze sequences for different taxa and their abundances without culturing any organisms has had a huge impact on the field,” Dr. Weinstock explained. In other words, NGS has removed the need for culturing by allowing the interrogation of genomic DNA that has been harvested directly from microbial communities.

Dr. Weinstock cited several relevant studies. One showed that classes of microbial communities differ from person to person. Another showed that children admitted to the emergency rooms with fevers of unknown origin often actually have viral infections. Yet another showed that the skin microbiome of diabetics differs from that of healthy subjects.


Solid-State Advantages

Shifting to more technical aspects of NGS, John Oliver, Ph.D., vice president of R&D at Nabsys, discussed the company’s semiconductor sequencing platform. In contrast to the optical detection typical of other sequencing platforms, semiconductor-based sequencing is based on the detection of hydrogen ions released during DNA polymerization.

“Completely semiconductor-based detectors are relatively rare at the moment in nucleic acid analysis,” said Dr. Oliver. “In contrast to the Ion Torrent platform, which is of course a short-read semiconductor sequencing technology, the Nabsys system is a long-read mapping technology.”

Dr. Oliver highlighted the myriad advantages of solid-state sequencing systems. These systems, he noted, can scale to encompass higher detector densities. Additional solid-state advantages include higher sensitivities, greater bandwidths, and lower costs.

Dr. Oliver also stressed situations in which long-read technology performs better than short-read technology: “The information content of short reads is insufficient in the context of large, repetitive genomes and hampers the detection of large structural variations.”

The Nabsys platform is based on a solid-state detector that is capable of sensing single DNA molecules as they transit the detector at high velocity (1 Mb/sec). In addition, the platform uses reads that are on the order of hundreds of kilobases. As a result,  said Dr. Oliver, the Nabsys platform can recover information about large structures.


Nabsys’ nano-channel detectors are capable of analyzing DNA at greater than 1M bases per second per detector.

NGS Library Quantification

Many scientists consider the most crucial steps in next-generation sequencing (NGS) library preparation to be the quantification of library molecules and the determination of fragment size range. Today’s NGS technologies require users to load a precise number of viable DNA library molecules onto the instrument to optimize data yield.

Performing a sequencing run with either too many or too few library molecules results in compromised data and sometimes no data at all, as well as wasted sample, expensive reagents, user time, and instrument time.

“In our research, we found that Bio-Rad’s Droplet Digital™ PCR (ddPCR) quantification is a more accurate and precise method for quality control of NGS libraries than conventional real-time PCR methods,” said Jason Bielas, Ph.D., who heads a laboratory in the Public Health Sciences Division at the Fred Hutchinson Cancer Research Center in Seattle. “Moreover, we have recently developed a ddPCR-based assay to concurrently measure both the absolute concentration and length of NGS libraries.”

The assay, named QuantiSize, exploits a linear correlation between the length of an amplified DNA molecule and the fluorescence amplitude produced in ddPCR.  This allows the user to calculate the size of an unknown DNA insert fragment.

“QuantiSize has enabled us to avoid the drawbacks of other independent quantification and size determination methods,” explained Dr. Bielas.


























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