A highly sensitive next-generation sequencing technique for detecting cancerous mutations
Unmet Need
Next-generation sequencing (NGS) methods have been widely adopted for both biological discovery and diagnostics in cancer patient care. In particular, the identification of new mutations or groups thereof arising within oncogenic pathways across cancer types is enabled by NGS and can potentially help stratify patients more precisely for optimal benefit from various therapies. However, the current state of NGS yields a sensitivity of 1 mutation/1x103 base pairs, whereas certain oncogenic driver mutations can occur at frequencies as low as 1 mutation/2x105 base pairs. It is thus necessary to vastly improve the sensitivity of NGS to identify driver mutations early in the oncogenic process to improve studying both the biology of cancer and prognosticating potential cancerous outcomes in patients.
Technology
Duke inventors have developed a highly sensitive NGS technique. This is intended to be used in biological investigations for identifying primordial oncogenic mutations, potentially to uncover insight into the oncogenic process and discover new therapeutic targets for cancer prevention. Specifically, the inventors apply Maximum Depth Sequencing (MDS), a method initially developed for bacterial genomics, to mammalian genomes which are 1000x larger in size and weight. By optimizing MDS for mammalian DNA, the inventors use the technique to probe urethane-induced Kras mutagenesis in mice. The process involves synthesizing unique barcodes onto a single strand of genomic region of interest, rounds of amplification and ultra-deep sequencing to identify bona fide mutations per barcode family. This has been demonstrated in mice exposed to urethane as the model carcinogen, inducing KrasQ61L mutations in the lungs immediately after exposure. Using the novel technique, the inventors demonstrate a detection sensitivity of one mutant per 2 million templates, several orders of magnitude better than conventional NGS. The inventors have yet to test other oncogenic mutations such as p53 or EGFR but it is clear the applicability of this method to other genes and organs.
Other Applications
This technology could also be used for cancer diagnostics for multiple tumor types, whereby early driver mutations on oncogenes are identified before therapeutic action is taken. This product could also become a direct-to-consumer genetic testing product.
Advantages
- Several orders of magnitude more sensitive than conventional NGS
- Adapts previously elaborated methodology and thus highly accessible
- Can be applied to many other genomic regions