Year: 2025
Type: preprint
Abstract: This protocol describes the automated extraction and SPRI of HMW DNA from metazoa samples intended for long-read sequencing using a combination of the Omega Bio-Tek E.Z.N.A.® Mollusc & Insect DNA kit, Sera-Mag™ SpeedBeads and the Thermo Fisher KingFisher™ Apex. It is effective for approximately several metazoa species covered by the Tree of Life Programme, such as isopods, amphipods, decapods, annelids and molluscs. This protocol has resulted in successful extractions for a number of species including Ligia oceanica, Titanethes albus, Oritoniscus flavus, Hyloniscus adonis, Oniscus asellus, Munidopsis polymorpha, Nephrops norvegicus, Willemoesia leptodactyla, Lumbricillus lineatus and the hemichordata Saccoglossus kowalevskii. The output of this protocol is HMW DNA, which - depending upon yield and genome size of the species - can be directed towards any of the Sanger Tree of Life HMW DNA Fragmentation protocols for LI or ULI PacBio sequencing. 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), 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
Authors: Amy Denton, Caroline Howard
Institution: Wellcome Sanger Institute
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4414684860
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