In recent years, next-generation sequencing approaches have generated unprecedented advances. With over 2,000 genes linked to at least one disease-relevant mutation, DNA sequencing has assumed increasingly important clinical relevance.
Nevertheless, multiple considerations still make it impractical to routinely sequence large numbers of eukaryotic genomes. Enrichment for specific regions of interest often becomes necessary, and targeted resequencing, which examines a limited number of genes within large populations, is emerging as a key approach, instrumental in our ability to unveil clinically and biologically relevant sequence variants.
Hanlee P. Ji, Ph.D., assistant professor in the department of medicine at Stanford University, and colleagues have developed several different approaches that allow targeted resequencing of genomic DNA and are useful for many applications such as the validation of mutations from cancer genomes, a topic that is still filled with challenges, partly due to the genetic heterogeneity found in clinical samples.
“This is one application for which targeted resequencing will be necessary and is unlikely to go away anytime soon,” explains Dr. Ji.
Another application is in clinical diagnosis, when multiple mutations in several genes are often linked to the same malignant tumor, but interpreting the clinical relevance of individual mutations is often a difficult task.
“I think what is practically going to happen is that gene subsets that have immediate clinical relevance and are clinically actionable will be quickly disseminated as diagnostic tests, and we already have started seeing that.”
Dr. Ji and colleagues recently published a targeted resequencing strategy that uses in-solution 80-mer oligonucleotides for capture and allows the analysis of gene subsets from the human exome. “This first-generation targeting technology is public and openly available for users.”
The Human OligoExome application is available at oligoexome.stanford.edu. Users can download the capture oligonucleotides to create their own assays independently of working with commercial sources.
“I think this is the first time that something like this has become available, on such a scale, for targeted resequencing,” explains Dr. Ji. The authors demonstrated the general robustness of this particular technology and revealed that it can resequence, with high sensitivity, genomic regions up to 1 megabase in size, when as little as tens of nanograms of target DNA is available.