Target enrichment for next-generation sequencing enables researchers to reduce the burden of sequence data analysis, obtain higher levels of statistical significance by examining more genomes and increase sequencing coverage depth. The goal of this STTR proposal is to develop and commercialize a novel technology capable of amplification-free megabase target sequence enrichment. This technology is highly differentiated from all other enrichment approaches currently on the market. Our method, MESA (Megabase Enrichment Sans Amplification), isolates large megabase-sized genomic DNA molecules in microfluidic drops and performs TaqMan PCR reactions to confirm the presence of the desired target sequence. The TaqMan identified drops are then sorted and collected to enrich for the target sequence prior to DNA sequencing. This approach is unique in that it is the only method that isolates intact megabase-sized fragments and does so without amplification of the DNA. The TaqMan PCR reaction in this procedure is used only to identify target sequences and not for the purpose of enriching through amplification itself. Furthermore, the TaqMan amplicons are removed from the enriched sample prior to sequencing. All other existing methods for target- enrichment fragment the DNA and enrich through PCR amplification or hybridization-based sequence capture. Both approaches suffer from various limitations including, lack of uniformity, specificity, depth of coverage or amplification induced bias/artifacs. Additionally, these approaches are often costly and time consuming, requiring the implementation of thousands of probes or primer sets. In this proposal, we aim to demonstrate enrichment of a megabase region of the human genome using a workflow that generates more uniform, specific and high coverage sequence data than is currently possible. The intellectual merits of the MESA approach stem from its ability to deliver a rapid, more robust and low-cost solution for targeted sequence enrichment. When combined with newer, long-read sequencing procedures, the MESA method also promises to aid in identifying new disease associated mutations that map to difficult to sequence regions of the genome. Commercialization of the MESA approach will generally enhance the utility of target sequence enrichment and further its adoption by researchers working in diverse areas of biology.
The vision of Torrent Bio, Inc. is to develop and commercialize a new target-enrichment tool for next- generation sequencing. This tool will be capable of uniform, specific and cost-effective enrichment of megabase size DNA fragments for sequencing. This capability will enable new investigations of genomes and the discovery of novel human disease associated genome alterations.
| Eastburn, Dennis J; Huang, Yong; Pellegrino, Maurizio et al. (2015) Microfluidic droplet enrichment for targeted sequencing. Nucleic Acids Res 43:e86 |
