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.