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


Type
Article
Change log
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.

Description
Keywords
HMW DNA extraction, genome assembly, long-read sequencing, tissue preservation, Animals, Benchmarking, DNA, Dimethyl Sulfoxide, Edetic Acid, High-Throughput Nucleotide Sequencing, Molecular Weight, Sequence Analysis, DNA
Journal Title
Gigascience
Conference Name
Journal ISSN
2047-217X
2047-217X
Volume Title
11
Publisher
Oxford University Press (OUP)
Sponsorship
Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z, 104640/Z/14/Z, WT207492)