Benchmarking Ultra-High Molecular Weight DNA Preservation Methods for Long-Read and Long-Range Sequencing

Authors

Hollis A. Dahn, University of Toronto
Jacquelyn Mountcastle, Rockefeller University
Jennifer Balacco, Rockefeller University
Sylke Winkler, Max Planck Institute of Molecular Cell Biology and Genetics
Iliana Bista, Wellcome Sanger Institute
Anthony D. Schmitt
Olga Vinnere Pettersson, Uppsala Universitet
Giulio Formenti, Rockefeller University
Karen Oliver, Wellcome Sanger Institute
Michelle Smith, Wellcome Sanger Institute
Wenhua Tan, Max Planck Institute of Molecular Cell Biology and Genetics
Anne Kraus, Max Planck Institute of Molecular Cell Biology and Genetics
Stephen Mac
Lisa M. Komoroske, University of Massachusetts Amherst
Tanya Lama, University of Massachusetts AmherstFollow
Andrew J. Crawford, Universidad de Los Andes, Colombia
Robert W. Murphy, University of Toronto
Samara Brown, Rockefeller University
Alan F. Scott, Johns Hopkins University School of Medicine
Phillip A. Morin, NOAA National Marine Fisheries Service Southwest Regional Office
Erich D. Jarvis, Rockefeller University
Olivier Fedrigo, Rockefeller University

Document Type

Article

Publication Date

1-1-2022

Publication Title

GigaScience

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.

Keywords

genome assembly, HMW DNA extraction, long-read sequencing, tissue preservation

Volume

11

DOI

10.1093/gigascience/giac068

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Rights

©The Author(s) 2022.

Comments

Archived as published.

Recommended Citation

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.; and Fedrigo, Olivier, "Benchmarking Ultra-High Molecular Weight DNA Preservation Methods for Long-Read and Long-Range Sequencing" (2022). Biological Sciences: Faculty Publications, Smith College, Northampton, MA.
https://scholarworks.smith.edu/bio_facpubs/315