Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing. (10th August 2022)

• Record Type: Journal Article
• Authors: Dahn, Hollis A; Mountcastle, Jacquelyn; Balacco, Jennifer; Winkler, Sylke; Bista, Iliana; Schmitt, Anthony D; Pettersson, Olga Vinnere; Formenti, Giulio; Oliver, Karen; Smith, Michelle; Tan, Wenhua; Kraus, Anne; Mac, Stephen; Komoroske, Lisa M; Lama, Tanya; Crawford, Andrew J; Murphy, Robert W; Brown, Samara; Scott, Alan F; Morin, Phillip A; Jarvis, Erich D; Fedrigo, Olivier
• Is Part Of: GigaScience. Volume 11(2022)
• Publication Date: 2022-08-10
• Subjects: long-read sequencing -- genome assembly -- tissue preservation -- HMW DNA extraction; Information storage and retrieval systems -- Research -- Periodicals; Biology -- Research -- Periodicals; Medical sciences -- Research -- Periodicals; Database management -- Periodicals; 570.285
• DOI: 10.1093/gigascience/giac068
• Languages: English
• ISSNs: 2047-217X

Abstract

Abstract: Background: Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. Results: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20–25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20–25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. Conclusion: We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70, 000 extant vertebrate species.