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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Dahn, HA; Mountcastle, J; Balacco, J; Winkler, S; Bista, I; Schmitt, AD; Pettersson, OV; Formenti, G; Oliver, K; Smith, M; Tan, W; Kraus, A; Mac, S; Komoroske, LM; Lama, T; Crawford, AJ; Murphy, RW; Brown, S; Scott, AF; Morin, PA; Jarvis, ED; Fedrigo, O

Published Date

  • August 10, 2022

Published In

Volume / Issue

  • 11 /

PubMed ID

  • 35946988

Pubmed Central ID

  • PMC9364683

Electronic International Standard Serial Number (EISSN)

  • 2047-217X

Digital Object Identifier (DOI)

  • 10.1093/gigascience/giac068

Language

  • eng

Conference Location

  • United States