Year: 2025
Type: preprint
Abstract: This protocol is for the manual extraction of HMW DNA from plant or fungi tissue samples from a variety of species intended for long-read sequencing using the Qiagen MagAttract HMW DNA extraction kit. This process is effective for approximately 60% of the plant species covered by the Tree of Life Programme, and the resulting yield of CCS data from PacBio sequencing has been good. This protocol is particularly useful for samples with limited tissue availability, as it ensures the maximum amount of HMW DNA can be extracted and recovered. 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 Pooling, HMW DNA Fragmentation: Opentrons® OT-2 for PacBio LI, HMW DNA Fragmentation: Diagenode Megaruptor® 3 for LI PacBio or HMW DNA Fragmentation: Covaris g-Tube for ULI PacBio. This protocol was adapted from Sanger Tree of Life HMW DNA Extraction: Manual Plant MagAttract v.4 to include an overnight elution in order to maximise the yield of DNA, as well as an updated pre-shear SPRI to improve recoveries of HMW DNA. Acronyms: HMW: high molecular weight SPRI: solid-phase reversible immobilisation LI: low input ULI: ultra-low input CCS: circular consensus sequencing
Authors: Graeme Oatley, Amy Denton, Anna Kovalevskaia, Caroline Howard
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W7084029219
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