Aug 07, 2019
High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing
- Rachael Workman1,
- Renee Fedak 2,
- Duncan Kilburn3,
- Stephanie Hao3,
- Kelvin Liu2,
- Winston Timp3
- 1Johns Hopkins University;
- 2Circulomics, Inc.;
- 3Johns Hopkins University, Department of Biomedical Engineering
- High molecular weight DNA extraction from all kingdoms
- TimpLab
External link: www.circulomics.com/support-nanobind
Protocol Citation: Rachael Workman, Renee Fedak , Duncan Kilburn, Stephanie Hao, Kelvin Liu, Winston Timp 2019. High Molecular Weight DNA Extraction from Recalcitrant Plant Species for Third Generation Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.4vbgw2n
Manuscript citation:
a. Y. Zhang, Y. Zhang, J. M. Burke, K. Gleitsman, S. M. Friedrich, K. J. Liu, and T. H. Wang, A Simple Thermoplastic Substrate Containing Hierarchical Silica Lamellae for High- Molecular-Weight DNA Extraction. Adv Mater (2016). PubMed PMID: 27862402
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 28, 2019
Last Modified: August 07, 2019
Protocol Integer ID: 25219
Keywords: Sequencing, third generation sequencing, nanopore, pacbio, DNA extraction, conifer, plants, redwood genome project, Nanobind, Circulomics, high molecular weight
Abstract
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+.
Attachments
Guidelines
Introduction
The assembly of high quality conifer genomes can benefit many fields of research from conservation and restoration efforts, to disease and stress studies, and evolutionary history. However, these tree genomes present unique assembly challenges; they are large (10-30+ Gb haploid), repetitive, and can have high ploidy. While long read sequencing, e.g. Oxford Nanopore, 10X, or PacBio, can greatly improve assembly contiguity, extracting large amounts of high quality, high molecular weight (HMW) DNA from adult trees presents a unique challenge. Although many extraction methodologies exist for recalcitrant plant species, most yield either DNA of quality “fit for PCR” and not for sensitive nanopore sequencing applications, or DNA too fragmented to obtain sequencing reads of sufficient length to improve assembly contiguity. Obtaining 60 kb+ and "nanopore clean" DNA places higher demands on sample extraction and preparation than existing methodology can provide in adult trees.
We have combined several techniques to develop HMW, "nanopore clean" DNA extraction methodologies from conifer species Sequoiadendron giganteum (giant sequoia) and Sequoia sempervirens (coast redwood) and generated sequence data on the Oxford Nanopore MinION. Our method integrates nuclei isolation and Nanobind DNA isolation (Circulomics) to improve purity and recovery 10-fold and reduce extraction time from 2-3 days to a single day. We also detail sequencing library preparation methodology and demonstrate extension of our methodology to Maize tissue.
Timing
Nuclei extraction: 2-3 hours
Nanobind gDNA extraction: 1-2 hours
Elution: 30 minutes
DNA relaxation (optional): overnight
Sequencing library preparation: 15 minutes - 2 hours
Oxford Nanopore Technologies Protocol Modifications
The ligation protocol was carried out as described for the LSK108 1D nanopore library preparation, with the exception of the following steps:
- Input to ligation protocol was 1.5 µg gDNA sheared to 8 kb with the Diagenode Megaruptor. Shearing to 10 kb with Covaris G-tubes (5000 x g for 00:01:00 each way) achieves similar results.
Note
Alternatively, for longer reads it is recommended not to shear and perform the ligation protocol as recommended, or include a size selection step with the Blue Pippin (Sage Sciences). Sequencing outcomes for all options are given in table below.
- End repair (NEBNext Ultra II) volumes were doubled over protocol recommendations (100 µL buffer, 14 µL enzyme), and the reaction was incubated at 20 °C for 00:20:00 and 65 °C for 00:20:00 .
Materials
MATERIALS
Liquid nitrogen
200 proof ethanol
ddH20
Potassium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P9541
0.5M EDTA solutionThermo Fisher ScientificCatalog #15575020
14 M ß-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148
Triton X-100 Merck MilliporeSigma (Sigma-Aldrich)Catalog #X100
Trizma® base Merck MilliporeSigma (Sigma-Aldrich)Catalog #T4661
Spermidine trihydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S2501
Spermine tetrahydrochloride Merck MilliporeSigma (Sigma-Aldrich)Catalog #S1141
Sucrose molecular biology gradeMerck MilliporeSigma (Sigma-Aldrich)Catalog #S0389
10 N NaOH Merck MilliporeSigma (Sigma-Aldrich)Catalog #72068
PVP 360K Merck MilliporeSigma (Sigma-Aldrich)Catalog #PVP360
Nanobind Plant Nuclei Big DNA Kit - Alpha Kit CirculomicsCatalog #SKU NB-900-801-01
Reagents
- 14 M ß-mercaptoethanol (Sigma-Aldrich, M3148-100ML)
- Triton X-100 (Sigma-Aldrich, X100-100ML)
- Trizma base (Sigma-Aldrich, T4661-100G)
- ddH20
- Potassium chloride (Sigma-Aldrich, P9541-500G)
- 0.5 M EDTA pH 8.0 (ThermoFisher, 15575020)
- Spermidine trihydrochloride (Sigma-Aldrich, S2501-5G)
- Spermine tetrahydrochloride (Sigma-Aldrich, S1141-5G)
- Sucrose, molecular biology grade (Sigma-Aldrich, S0389-1KG)
- 10 N NaOH (Sigma-Aldrich, 72068-100ML)
- PVP 360K (Sigma-Aldrich, PVP360-100G)
- 200 proof ethanol
- Liquid nitrogen
Nanobind Plant Nuclei Big DNA Kit - Alpha Kit (Circulomics Inc)
- Nanobind disks
- Proteinase K
- RNase A
- Buffer PL1 - Lysis/Binding buffer
- Buffer PW1 Concentrate - Wash buffer concentrate
- Buffer EB - Elution buffer
Equipment
- Refrigerated centrifuge
- Paintbrushes
- Miracloth (Millipore Sigma, 475855-1R)
- Conical vials (15 mL and 50 mL)
- 250 mL capped bottle
- 100 mL beaker
- End over end mixer (optional)
- Magnetic stir plate + stir bars
- pH meter or strips
- Fume hood
- Funnel
- NanoDrop and/or Qubit Fluorometer (Thermo Fisher)
- ThermoMixer (Eppendorf)
- HulaMixer (Thermo Fisher)
Reagent setup
HB (homogenization buffer) stock 10X (100 mL)
| 1.21 g | Trizma Base | |
| 5.96 g | KCL | |
| 20 mL | 0.5 M EDTA | |
| 0.255 g | Spermidine | |
| 0.348 g | Spermine | |
| Fill to 100 mL | ddH20 |
Adjust pH to 9-9.4 with 10M NaOH Drops. Can store at 4 °C in a glass bottle for up to 1 year
HB 1X solution (1000 mL)
| 100 mL | 10X HB | |
| 171.2 g | Sucrose | |
| Fill to 700 mL | ddH20 (stir until dissolved) |
Bring to final volume of 1L. Can store at 4 °C in glass bottle for 3 months.
Triton X-100 (20% vol/vol, 100 mL)
| 20 mL | Triton X-100 | |
| 10 mL | 10X HB | |
| 17.15 g | Sucrose | |
| Fill to 60 mL | ddH20 (stir until dissolved) |
Bring to final volume of 100 mL Can store at 4 °C in a glass bottle for up to 1 year.
Prepare day of isolation:
NIB (nuclear isolation buffer)
Make 10 mL NIB per gram of tissue, plus an additional 50 mL for washes. Example recipe for 5 g of tissue:
| 97.5 mL | 1X HB | |
| 2.5 mL | Triton X-100 mix | |
| 250 μl | ß-mercaptoethanol | |
| 1 g | PVP |
Stir until mixed
Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.
Before start
Nuclei Isolation
Nuclei Isolation
Grind 1 g of tissue, preferably fresh or snap frozen, into fine powder in liquid nitrogen with a mortar and pestle. Immediately transfer ground tissue to capped 250 mL bottle containing 10 mL NIB.
Cap bottle and attach to end over end mixer, rotating at max speed for 00:15:00 at 4 °C .
Grind 1 g of tissue, preferably fresh or snap frozen, into fine powder in liquid nitrogen with a mortar and pestle.
Immediately transfer ground tissue to capped 250 mL bottle containing 10 mL NIB.
Cap bottle and attach to end over end mixer, rotating at max speed for 00:15:00 at 4 °C .
Note
Alternatively, lay bottles on their side on a shaker (150 rpm), or transfer ground tissue to a beaker capped with foil and mix on stir plate with stir bar at 4 °C for 00:15:00 .
Using a funnel, gravity filter homogenate through 5 layers of Miracloth into a 50 mL conical tube. Cap tube and centrifuge at 4 °C for 00:20:00 .
Using a funnel, gravity filter homogenate through 5 layers of Miracloth into a 50 mL conical tube.
Cap tube and centrifuge at 4 °C for 00:20:00 .
Note
Speed of centrifugation is dependent upon size of genome:
For larger genomes such as redwood (30 Gb), we spun down at 1900 x g .
For smaller genomes like walnut (1 Gb) we spun down at 2900 x g .
For maize (2.5 Gb) was spun down at 2500 x g .
Decant the supernatant and add 1 mL cold NIB to pellet. Resuspend pellet with paint brush pre-soaked in NIB.
Decant the supernatant and add 1 mL cold NIB to pellet.
Resuspend pellet with paint brush pre-soaked in NIB.
Note
Pipetting up and down with wide bore tips works for some species (for example maize) but for many species the pellet is too sticky to allow for sufficient resuspension by pipet.
Transfer 1 mL nuclei suspension to a 15 mL conical.
Bring volume up to 15 mL with ice-cold NIB. Centrifuge at 4 °C for 00:10:00 .
Bring volume up to 15 mL with ice-cold NIB.
Centrifuge at 4 °C for 00:10:00 .
If after centrifugation supernatant is clear, decant supernatant and take pellet into step 7.
If coloration remains in the supernatant after centrifugation, dispose of supernatant and resuspend nuclei pellet in NIB, bringing volume up 10 mL - 15 mL , and go to step #5.2 .
Note
For conifer tissue it was important to repeat this step 3-5X; however, in maize tissue subsequent washes have been unnecessary.
Remove supernatant and resuspend in 1 mL 1X HB.
At this point, you can either snap freeze nuclei or proceed to lysis.
- Please select 'Snap Freezing' or 'Lysis' to proceed.
Snap Freezing1 step
Snap Freezing
Snap Freezing
Spin down your nuclei suspension in a 1.5 mL tube at 5000 x g - 7000 x g for 00:05:00 , remove supernatant and snap freeze in liquid nitrogen, then store at -80 °C .