High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
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/HighMolecular_Weight_DNA_Extraction_from_Recalcitrant_Plant_Species_for_Third_Generation_Sequencing-Version_1-_Protocol_Exchange.pdf?1529318652
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Protocol - Version 1 protex.2018.059 - Version 1 The protocol has been modified since it was originally published. This is the original version. Step 8 has been corrected. In the original version: ""8. At this point, you can either snap freeze nuclei or proceed to lysis. If snap freezing, spin down your nuclei suspension in a 1.5 mL tube at 5000-7000 x g for 5 minutes, remove supernatant and snap freeze in liquid nitrogen, then store at -80 °C. Otherwise proceed to Nanobind-assisted DNA purification." it should read: "8. At this point, you can either snap freeze nuclei or proceed to lysis. If snap freezing, spin down your nuclei suspension in a 1.5 mL tube at 5000-7000 x g for 5 minutes, remove supernatant and snap freeze in liquid nitrogen, then store at -80 °C. If proceeding to Nanobind-assisted DNA purification, spin down your nuclei suspension in a 1.5 mL tube at 5000-7000 x g for 5 minutes, remove supernatant and proceed with step 1 of the Nanobind-assisted DNA purification section below." 18 June 2018
HB recipe lets adjust the pH to 9-9.4 while troubleshooting warns that HB pH should be 8.5 to 9. Which is true? Thanks for your feedback. (submitted on behalf of Stephane Plaisance)
Hi, Do you autoclave or filter sterilize the buffers given in the protocol. Thank you, Ramesh
Posted 27 Apr, 2018
High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
Posted 27 Apr, 2018
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/HighMolecular_Weight_DNA_Extraction_from_Recalcitrant_Plant_Species_for_Third_Generation_Sequencing-Version_1-_Protocol_Exchange.pdf?1529318652
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Our nuclei isolation on Phaseolus vulgaris made use of a small paintbrush, with a bristle length of 0.5-1.0 cm. and a width of around 0.5cm. Using a brush with sort bristles helped to resuspend the nuclei pellet.
HB recipe lets adjust the pH to 9-9.4 while troubleshooting warns that HB pH should be 8.5 to 9. Which is true? Thanks for your feedback. (submitted on behalf of Stephane Plaisance)
Hi, Do you autoclave or filter sterilize the buffers given in the protocol. Thank you, Ramesh
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Greg Perry
replied on 10 May, 2019
Our nuclei isolation on Phaseolus vulgaris made use of a small paintbrush, with a bristle length of 0.5-1.0 cm. and a width of around 0.5cm. Using a brush with sort bristles helped to resuspend the nuclei pellet.