Single molecule sequencing requires optimized sample and library preparation protocols to obtain long-read lengths and high sequencing yields. Numerous protocols exist for the extraction of DNA from plant species, but the genomic DNA from these extractions is either too low yield, of insufficient purity for sensitive sequencing platforms, e.g. nanopore sequencing, too fragmented to achieve long reads, or otherwise unattainable from recalcitrant adult tissue. This renders many plant sequencing projects cost prohibitive or methodologically intractable. Existing protocols are also labor intensive, taking days to complete. Our protocol described here yields micrograms of high molecular weight gDNA from a single gram of adult or seedling leaf tissue in only a few hours, and produces high quality sequencing libraries for the Oxford Nanopore system, with typical yields ranging from 3-10 Gb per R9.4.1 flowcell and producing reads averaging 5-8 kb, with read length N50s ranging from 6-30 kb depending on the style of library preparation (details in sequencing outcomes section), and maximum lengths extending up to 200 kb+.
Editor note, 18 June 2018: Step 8 of the procedure has been updated. The original version of the protocol can be found "here":http://www.nature.com/protocolexchange/system/uploads/6879/original/High_Molecular_Weight_DNA_Extraction_from_Recalcitrant_Plant_Species_for_Third_Generation_Sequencing_-Version_1-_Protocol_Exchange.pdf?1529318652