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Method Article
High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
Rachael Workman
Timp Lab, Johns Hopkins University Department of Biomedical Engineering
Corresponding Author
Winston Timp
Timp Lab, Johns Hopkins University Department of Biomedical Engineering
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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
Keywords
Sequencing, third generation sequencing, nanopore, pacbio, DNA extraction, conifer, plants, redwood genome project, Nanobind, Circulomics, high molecular weight
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- supplement0.pdf
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
View 1 reply
Bronwen Dekker
commented on 17 June, 2019
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)
Ramesh Vetukuri
commented on 25 February, 2020
Hi, Do you autoclave or filter sterilize the buffers given in the protocol. Thank you, Ramesh
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This is a preprint. It has not completed peer review.
Method Article
High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
Rachael Workman
Timp Lab, Johns Hopkins University Department of Biomedical Engineering
Corresponding Author
Winston Timp
Timp Lab, Johns Hopkins University Department of Biomedical Engineering
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 27 Apr, 2018
Community comments: 3
Metrics
Comments: 3
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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
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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.
Bronwen Dekker
commented on 17 June, 2019
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)
Ramesh Vetukuri
commented on 25 February, 2020
Hi, Do you autoclave or filter sterilize the buffers given in the protocol. Thank you, Ramesh
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.