Sanger Tree of Life HMW DNA Extraction: Modified Omega Bio-Tek E.Z.N.A.® v1

This protocol describes the manual extraction and SPRI of HMW DNA from fresh frozen jellyfish samples intended for long-read sequencing using a combination of the Omega Bio-Tek E.Z.N.A.® Mollusc & Insect DNA kit and Sera-Mag™ SpeedBeads. It is effective for approximately 50 to 60% of jellyfish species covered by the Tree of Life Programme for the Aquatic Symbiosis Genomics (ASG) project. This protocol has resulted in successful extractions for a number of species including Mastigias papua, Mastigias albipunctatus, Rhopilema esculentum, Catostylus mosaicus, Cassiopea ornata, Cassiopea xamachana, Mertensia ovum, Bolinopsis mikado, Pseudorhiza haeckeli, Aurelia spp. and Pelagia noctiluca. The output of this protocol is HMW DNA, which depending upon yield and genome size of the species, can be directed towards either HMW DNA Fragmentation: Diagenode Megaruptor®3 for LI PacBio or HMW DNA Fragmentation: g-Tube for ULI PacBio. This protocol was developed through R&D by the Tree of Life Core Laboratory; it was primarily adapted from the Omega Bio-Tek E.Z.N.A.® Mollusc & Insect DNA kit protocol by combining the initial lysis and precipitation (steps 1 to 10) with reduced incubation temperatures, omission of vortexing and the addition of a bead-based extraction using Sera-Mag™ SpeedBeads, as utilised in the Sanger Tree of Life HMW DNA Extraction: Automated Plant Organic HMW gDNA Extraction (POE) protocol. Acronyms HMW: high molecular weight LI: low input ULI: ultra-low input