Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.
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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
Gigascience 11 (-) - [2022-08-10; online 2022-08-11]
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. 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. 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.
NGI Long read [Technology development]
NGI Uppsala (Uppsala Genome Center) [Technology development]
National Genomics Infrastructure [Technology development]
PubMed 35946988
DOI 10.1093/gigascience/giac068
Crossref 10.1093/gigascience/giac068
pii: 6659719
pmc: PMC9364683