Abstract
Abstract High-quality UHMW DNA is key to success in optical mapping of long genomic DNA using Bionano Genomics Saphyr® system for de novo genome assembly and structural variation detection. Ideally, ~100 mg of tissue is frozen immediately upon collection using liquid nitrogen or dry ice, and stored at -80°C before DNA isolation. Practically, tissue amounts may be limited and collection often occurs at sites where low temperature preservation for storage and shipping may be unavailable. Here we present a novel method to isolate UHMW DNA from 10 mg of either freshly frozen or room temperature preserved animal and human tissue for subsequent enzymatic labeling using direct labeling and staining (DLS) to generate Bionano maps. With a TissueRuptor, 10 mg tissue is homogenized followed by a three-step purification process before embedding the purified nuclei in agarose gel plugs. This process takes less than 3 hours for a batch of 6 samples. Using this method, we have successfully isolated high quality UHMW DNA from various tissue types of rat and human including solid tumors. The method is very attractive compared to other protocols because: 1) tissue preservation, shipping and handling can be done at room temperature, 2) uses very small amount of tissue - possible application for rare samples and human tissue biopsy tests, and 3) the length and quality of the resulting single molecules generate high quality optical maps for genome finishing and SV calling. Citation Format: Yang Zhang, James Broach. A novel method for isolating high-quality UHMW DNA from 10 mg of freshly frozen or liquid-preserved animal and human tissue including solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5125.
Published Version
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