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Mar 1, 2012 (Vol. 32, No. 5)

NGS Advances Spawn Novel Challenges

  • Improving Sample Prep

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    Multiplexed methylated DNA immunoprecipitation (MeDIP) can now be performed with less than 10 ng of DNA input using bead-based cleanup, according to Bioo Scientific. Methylated DNA immunoprecipitation is a useful immuno-capturing approach for accurate detection of methylated DNA. Enriched methylated DNA in IP fractions can be used to assess genome-wide methyl profiling. It’s basically ChIP-Seq with more epigenetic information, the firm notes.

    Once samples are collected—whether from a clinic or a basic research lab—they cannot be loaded directly into a sequencing machine, of course. One issue is that the DNA of interest needs to be relatively free of irrelevant DNA and other impurities. A second issue is that the samples must be rendered ready for a specific sequencing machine.

    “The instrument companies utilize varied adapters and different ligation strategies, making it difficult for users to prepare a one size fits all sample,” explains Masoud Toloue, Ph.D., director of scientific research at Bioo Scientific. “Unfortunately, I don’t see that changing in the future.”

    Rubicon Genomics currently offers kits to prepare clinical samples for sequencing via the Illumina platform and is working toward compatibility with the Life Technologies platform.

  • For a review of a paper published about Life Technologies' Ion Torrent sequencing technology, click here.

  • Dr. Langmore notes that sample-preparation procedures acceptable for research samples are usually inadequate for clinical samples.

    Researchers starting with microgram amounts of homogeneous DNA can afford to lose 99% of it during preparation, but that is not an option for people aiming to sequence sub-nanogram amounts of fetal DNA or tumor DNA mixed with nanogram amounts of maternal or nontumor DNA.

    Furthermore, short segments of free-floating DNA found in plasma samples are best handled differently than formalin-fixed cells prepared for pathology analysis, in which the DNA is preserved in much larger pieces but is damaged in the fixing process.

    Rubicon’s ThruPLEX-FD kit is designed to retain >90% of target DNA from plasma and formalin samples in a one-tube, two-hour, three-step process compatible with the high-throughput format of Illumina sequencers. Dr. Langmore believes that forthcoming kits targeting individual sample types—for example, a plasma-specific kit—can further simplify the process to two steps and less than one hour.

    At Bioo Scientific, Dr. Toloue is tackling the related challenge of developing genome-scale methods for profiling DNA methylation. 5´ methylation of cytosine bases is a general mechanism for suppressing gene expression and is thus of great interest to both basic researchers and clinicians.

    However, among other problems, the DNA polymerases commonly used in amplification and sequencing reactions do not recognize uracils introduced via bisulfite conversion of unmethylated cytosines, which allows these cytosines to be distinguished from methylated ones.

    Dr. Toloue will discuss advances in methods to capture whole-genome methylation data and will compare the methylomes of differentiated and undifferentiated cells. “At this stage we are examining research samples,” he says. “The results of this study, however, expand into development of induced pluripotent stem cells as clinical replacements to human embryonic stem cells.”

  • Translating Data into Clinical Advice

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    The current version of Existence Genetics’ Nexus DNA chip reportedly tests about 10,000 loci, though this number will increase as additional disease-linked genetic variants are discovered.

    Many researchers consider valid and affordable clinical applications of whole-genome analysis to be at least a couple of years away. Existence Genetics, however, already offers assessment of clients’ genetic predisposition toward hundreds of diseases.

    While whole-genome sequencing is not yet a cost-effective way of gathering clinically relevant data, Existence’s Nexus DNA Chip tests genetic loci linked to hundreds of diseases. Brandon Colby, M.D., the firm’s founder and CEO, describes it as “an aggregation of all known loci associated with a phenotype—an increased or decreased risk of disease—as discovered by third-party research studies over the last three decades.”

    The current version of the Nexus chip tests about 10,000 loci, though this number will increase as additional disease-linked genetic variants are discovered.

    In addition to partnering with medical clinics, Existence offers its services through outlets such as the Equinox Fitness chain of health clubs, which offer to estimate clients’ relative risk of developing such conditions as arthritis and osteoporosis.

    “If somebody has a very high risk of knee arthritis, then trainers tailor a program, such as cardiovascular exercise, that has low impact on the knees,” Dr. Colby explains. “Instead of running on the treadmill, they’re focusing on the elliptical, or on the bike, or on swimming.”

    Similarly, women whose results indicate a predisposition toward osteoporosis are given extra motivation to strengthen their bones via resistance training.

    For his part, Dr. Diehl worries that the promise of NGS advances will be distorted by the “hype cycle” of media organizations and profit-driven companies. Yet he remains optimistic that the public will ultimately embrace the ambitious long-term studies needed to shed further light on the genetic basis of complex diseases.

    He cites a recent conversation with Danish researchers who have had good success recruiting subjects for pain studies.

    “Who wants to volunteer for a study of pain?” Dr. Diehl wonders. “Good grief! But it sounds like the culture in Denmark is that people feel a responsibility that, if I and my family are going to benefit from biomedical research, I should be responsible for participating in this research. It’s not fair for me to just sit on the sidelines…and then gain the benefits of the new medicines.”


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