Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing

Hollis A. Dahn, Jacquelyn Mountcastle, Jennifer Balacco, Sylke Winkler, Iliana Bista, Anthony D. Schmitt, Olga Vinnere Pettersson, Giulio Formenti, Karen Oliver, Michelle Smith, Wenhua Tan, Anne Kraus, Stephen Mac, Lisa M. Komoroske, Tanya Lama, Andrew J. Crawford, Robert W. Murphy, Samara Brown, Alan F. Scott, Phillip A. MorinErich D. Jarvis, Olivier Fedrigo

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Article numbergiac068
JournalGigaScience
Volume11
DOIs
StatePublished - 2022

Keywords

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

ASJC Scopus subject areas

  • General Medicine

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