Research Project
9778132
Summary Claremont BioSolutions, LLC (CBIO) The introduction of long-read sequencing and mapping applications is set to change the field of cancer research by allowing unparalleled insight into the complex structure of tumor genomes. With the ability to sequence large spans of DNA that are 10s-100s of kb in size these technologies will offer more accurate de novo assembly, resolve highly repetitive regions, and allow identification of large structural variants in tumor genomes. As cancer researchers move to these platforms they will face new hurdles as effective sample preparation of ultra-long DNA (>250 kb) remains a limiting step. Current methods of DNA extraction, including spin column and magnetic bead applications, significantly shear ultra-long DNA. Older, albeit effective, agarose plug digestion techniques are difficult, time-consuming and highly variable. New commercial products have been introduced that can isolate ultra-long DNA but are restricted in use to bacteria, tissue culture cells, or fresh blood cells, and are not usable with clinically relevant solid tumor tissue samples. To overcome this challenge, Claremont BioSolutions is developing a prototype technology that combines gentle and rapid tissue dissociation with a novel binding matrix to isolate high quality/high quantity ultra-long DNA in <20 minutes. The method reduces DNA shearing and tangling and is compatible with small quantities of solid tissue. In Aim 1, we shall use the combined approach to demonstrate isolation of ultra-long DNA (>250 kb) from matched normal and tumor tissue samples and analyze length and quality. In Aim 2, we shall validate compatibility of the isolated ultra-long DNA with third generation long-read sequencing and mapping technologies and analyze the sequence data to demonstrate improved long-read sequencing over existing DNA extraction methods. Ultimately, the goal will be to integrate this miniature technology into an automated platform to provide a rapid, cost effective, and reproducible method to isolate DNA from solid tissues for improved downstream analysis of tumor samples.
