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Jun 15, 2010 (Vol. 30, No. 12)

Next-Generation Sequencing Moves to Next-Next Level

As Technology Proves Its Mettle and Advances Expand Usage, Applications Are Proliferating

  • Computational Challenges

    One of the major challenges when examining whole-genome sequences is that particularly novel sequence insertions, by definition, are missing from the reference human genome, and their omission can lead to incomplete genome analysis.

    Furthermore, many previous studies on structural variation avoid chromosomal repeat regions, despite the fact that these repeats constitute about 40% of the human genome. In addition to the large amount of information that can be missed by not analyzing these regions, it is also known that they harbor structural alterations more often than the rest of the chromosome.

    “I think it is important to understand complex repeat regions in the chromosomes and to improve the algorithmic knowledge, because if we don’t have the right methods, with the right mathematical tools behind it, it is difficult to arrive at the right conclusions,” said S. Cenk Sahinalp, Ph.D., professor of computing science and director of the laboratory for computational biology at Simon Fraser University.

    To define the location and content of novel genomic sequence insertions, Dr. Sahinalp and collaborators recently developed and validated NovelSeq, a computational framework that uses sequencing data generated by next-generation sequencing platforms. By using this tool, the authors unveiled recurrent structural variants in several cell lines and cancer tissues from patients. “I really encourage researchers not to ignore repeat regions because there is a lot of interesting data in them. The lack of appropriate algorithmic tools can really hurt the progression of the field,” he emphasized.

    Dr. Sahinalp and collaborators are currently working on making the software even more user friendly and developing a web interface to enable more users to analyze and interpret their data.

  • Mechanistic Details

    An important application of next-generation sequencing methods is to understand the mechanism of action of small molecule drugs. An informative model system is provided by the budding yeast, which is inexpensive, easy to manipulate, and represents a well-characterized and powerful genetic tool. In addition, a complete deletion collection, where every single gene from the genome has been replaced with a unique barcode identifier, is available. It allows massively parallel experiments to be conducted by growing all the mutants simultaneously.

    The consequence of a specific perturbation such as a therapeutic agent can be examined by PCR amplifying the barcode sequence and hybridizing the products to a barcode microarray that is complementary to the unique identifiers. By using this approach, Corey Nislow, Ph.D., and Guri Giaever, Ph.D., both assistant professors in the department of molecular genetics at the University of Toronto, and collaborators, recently conducted massively parallel experiments and revealed that, in addition to the ability to interrogate 6,200 different mutants in one experiment, multiplexing is also associated with a great reduction in costs.

    “In a sense, we are using next-generation sequencing as a simple and powerful molecular counter,” revealed Dr. Nislow. However, the quantity of data that is generated is not the only benefit, and this approach is attractive for several other reasons. “Instead of a time point, it is now possible to perform an entire drug titration, generate a complex time course, or even look at treatment combinations.” This aspect is significant, because it is becoming increasingly clear that most therapeutic agents are more effective in combination than as single agents.

    In addition, Dr. Nislow’s group collaborates with Jason Moffat, Ph.D.’s lab at the University of Toronto to conduct a similar type of screening that uses mammalian cells in which, instead of deletions, gene function is knocked down by shRNA. “We are taking what we are learning from yeast and applying the findings to mammalian cell-based assays,” explained Dr. Nislow.

    As these recent developments reveal, next-generation sequencing is witnessing exciting times. The ability to survey the extensive inter-individual genomic variations, understand the complex interplay between genetic and epigenetic modifications, and dissect the response to therapeutic agents are only some of the applications that place this technology at the forefront of clinical and research laboratories, where it promises important prophylactic, diagnostic, and therapeutic benefits.

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