Apr 04, 2022
High Molecular Weight DNA extraction for long-read sequencing v.1
- Kanae Nishii1,2,
- Michael Möller1,
- Michelle Hart1
- 1Royal Botanic Garden Edinburgh;
- 2Kanagawa University
Protocol Citation: Kanae Nishii, Michael Möller, Michelle Hart 2022. High Molecular Weight DNA extraction for long-read sequencing v.1. protocols.io https://dx.doi.org/10.17504/protocols.io.bempjc5n
Manuscript citation:
Nishii K, Hart M, Kelso N, Barber S, Chen Y-Y, Thomson M, Trivedi U, Twyford A D, Möller M (2022) The first genome for the Cape Primrose Streptocarpus rexii (Gesneriaceae), a model plant for studying meristem-driven shoot diversity. Plant Direct (In prep)
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: April 05, 2020
Last Modified: April 04, 2022
Protocol Integer ID: 35215
Keywords: DNA extraction, high molecular weight DNA, long read sequencing, Streptocarpus, Qiagen Genomic-tip
Abstract
Third generation long-read sequencing requires high quality DNA. Here, we developed a protocol for the extraction of high quality, high molecular weight(HMW) DNA from plants. This protocol was developed and optimized forStreptocarpus, a plant possessing high amounts of secondary metabolic compounds, such as polysaccharides. The extracted DNA was used for PacBio Sequel II continuous long reads (CLR) and Oxford Nanopore Technologies PromethION sequencing with the resulting reads averaging > 25,000 base-pairs.
References
Nishii K, Möller M, Hart M (2019) DNA extraction protocol for long read sequencing; DNA extraction for state-of-the-art sequencing. Botanic Stories: 30792. https://stories.rbge.org.uk/archives/30792
Gunter L (2015) Populus nuclear DNA purification using the QIAGEN Genomic-tip 100/G.
PacBio Sample Net (2015) Preparing Arabidopsis genomic DNA for size-selected ~20 kb SMRTbell™ Libraries. Access year: 2018. https://www/pacb.com/wp-content/uploads/2015/09/Shared-Protocol-Preparing-Arabidopsis-DNA-for-20-kb-SMRTbell-Libraries.pdf
Souza HA, Muller LA, Brandao RL, Lovato MB (2012) Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado. Genet Mol Res 11: 756-764
Guidelines
This is the protocol used for Streptocarpus long-read whole genome sequencing with PromethION (Oxford Nanopore Technologies) and Sequel II CLR (Pacific Biosciences).
The protocol described below is optimized for 30 g of starting material (i.e. fresh leaves) of Streptocarpus.
Materials
MATERIALS
Proteinase KQiagenCatalog #19131
RNaseAThermo FisherCatalog #12091021
Genomic-tip 100/GQiagenCatalog #10243
RNaseA/T1 MixThermo FisherCatalog #EN0551
Buffer G2QiagenCatalog #1014636
Buffer QBTQiagenCatalog #19054
Buffer QCQiagenCatalog #19055
Buffer QFQiagenCatalog #19056
Cell strainer 40 µmCorningCatalog #431750
Cell strainer 100 µmCorningCatalog #431752
*See above for specific materials` details
Equipments
Fume hood
Water bath
Heat block
Centrifuges for 50 ml Falcon tubes
Centrifuges for 1.5 ml and 2.0 ml Eppendorf tubes
Spectrophotometer: Nanodrop or DeNovix
Fluorometer: Qubit or DeNovix
Pipetman (1 ml, 200 μl, 20 μl)
500 ml glass beakers
Pestle and mortar (1 set per 1-2 g of fresh leaf material)
Reagents & Consumables
50 ml Falcon tube
15 ml Falcon tube
2 ml Eppendorf tube
1.5 ml Eppendorf tube
2 ml LoBind Eppendorf tube
1ml Pipetman tips
1ml Wide orifice tips (or cut off tip ends)
200 μl Pipetman tips
20 μl Pipetman tips
Nylon mesh 100 µl mesh size (e.g. Corning cell strainer*)
Nylon mesh 40 µl mesh size (e.g. Corning cell strainer*)
QIAGEN Genomic-tip 100/G columns*
QIAGEN Buffer G2*
QIAGEN Buffer QBT*
QIAGEN Buffer QC*
QIAGEN Buffer QF*
Nuclei isolation buffer (NIB) base solution1
Nuclei isolation buffer (NIB)2
NIB base-Triton mix (10% Triton X-100 in NIB base solution)3
Sorbitol buffer base solution (2 ×)4
Sorbitol buffer5
Proteinase K*
RNaseA6 or RNase A/T1 Mix*
Liquid Nitrogen
Isopropanol
3 M NaOAc (pH 5.2)
70% ethanol
Polyvinyl polypyrrolidone (PVPP)
PVP-40
β-Mercaptoethanol
4 M spermidine (store at −20 °C)7
1 M Tris-HCl pH 8.0
500 mM EDTA pH 8.0
Sucrose
KCl
1. Nuclei isolation buffer (NIB) base solution
Final concentrationsof NIB base solution
Tris-HCl pH 8.0 10 mM
EDTA pH 8.0 10 mM
KCl 100 mM
Sucrose 500 mM
To prepare 1 L stock of NIB base solution:
Add the chemicals below in >1 L autoclavable bottle.
10 ml 1 M Tris-HCl pH 8.0
20 ml 500 mM EDTA pH 8.0
7.4 g KCl
172 g Sucrose
Add distilled water to makeup 1 L
Mix until sucrose and KCl are dissolved
Autoclave and store at 4 ºC
2. Nuclei isolation buffer (NIB)
Note: Prepare 200 ml NIB fresh on the day of DNA extraction
Final concentrations of NIB
Tris-HCl pH 8.0 10 mM
EDTA pH 8.0 10 mM
KCl 100 mM
Sucrose 500 mM
Spermidine 4 mM
β-mercaptoethanol 0.1%
To prepare 200 ml NIB
Pre-cool a 500ml beaker on ice and add:
200 ml NIB base solution
200 μl β-mercaptoethanol
200 μl 4 M spermidine
Keep NIB cooled on ice.
3. NIB base-Triton mix (10% Triton X-100 in NIB base solution)
In a 50 ml Falcon tube, add:
5 ml Triton X-100
45 ml NIB base solution
Gently mix
It can be stored at 4 ºC
Note: Pour Triton X-100, as it is a sticky liquid and pipetting does not work.
4. Sorbitol buffer base solution (2 × stock)
Final concentrations of 2 × Sorbitol buffer base solution
Tris-HCl pH 8.0 200 mM
EDTA pH 8.0 10 mM
Sorbitol 700 mM
To prepare 500 ml of Sorbitol buffer base solution (2 × stock)
Ina 500 ml autoclavable glass bottle, add:
100 ml 1 M Tris-HCl pH 8.0
20 ml 500 mM EDTA pH 8.0
128 g Sorbitol
Add distilled water to makeup 1 L
Mix to dissolve sorbitol
Autoclave and store at 4 °C
5. Sorbitol buffer
Final concentrations of Sorbitol buffer
Tris-HCl pH 8.0 100 mM
EDTA pH 8.0 5 mM
Sorbitol 350 mM
β-mercaptoethanol 1%
PVP-40 1%
Prepare 60 ml Sorbitol buffer on the day of DNA extraction
To prepare 60 ml Sorbitol buffer
Prepare two 50 ml Falcon tubes in a rack and add the following chemicals in each tube:
15 ml Sorbitol buffer base solution
300 μl β-mercaptoethanol
0.3 g PVP-40
Add distilled water to makeup 30 ml
Note: Increasing the concentration of Sorbitol (100 mM Tris-HCl pH8.0; 5 mM EDTA pH8.0; 700 mM sorbitol) worked well for Streptocarpus.
Reference: Souza HA, Muller LA, Brandao RL, Lovato MB (2012) Isolation of high quality and polysaccharide-free DNA from leaves of Dimorphandra mollis (Leguminosae), a tree from the Brazilian Cerrado. Genet Mol Res 11: 756-764
6. RNase A
We recommend to incubate RNase A at 95 °C for 5 minutes in a thermocycler prior to use.
7. 4 M spermidine
Elute spermidine powder in distilled water to makea4 M solution. The molecular weight of spermidine is 145.25 g/mol and thus e.g. dissolve 5 g spermidine powder in approx. 8.6 ml distilled water. Divide dissolved spermidine solution into several 1.5 ml Eppendorf tubes. Store the tubes at −20 °C.
Safety warnings
Perform experiment in the fume hood for the steps using liquid nitrogen and β-mercaptoethanol.
Before start
Prepare Nuclei isolation buffer base solution and Sorbitol buffer base solution.
Cultivating fresh plant material > 100 g is recommended.
Before starting
Before starting
Prepare 200 ml Nuclei isolation buffer (NIB) and leave on ice.
On ice
Prepare 20 ml NIB base-Triton mix and leave on ice.
On ice
Prepare 60 ml Sorbitol buffer.
Prepare liquid nitrogen in a suitable dewar. Place mortar and pestle in fume hood.
Prepare six 50 ml Falcon tubes on ice and place 100 μm gauge cell strainers (Nylon Meshes) on top.
On ice
Prepare the other set of six 50 ml Falcon tube layered with 40 μm gauge cell strainers, leave on ice.
On ice
Tissue grinding
Tissue grinding
Pre-cool the pestle and mortar with liquid nitrogen.
Cut 1-2g of fresh leaf tissue from living plants and place in liquid nitrogen to shock-freeze.
Add frozen tissue to pre-cooled mortar held in a Styrofoam mortar holder.
Add some liquid nitrogen and grind carefully with pestle as the liquid nitrogen evaporates.
Once the liquid nitrogen has evaporated, grind more vigorously for 30 seconds, until the material is a fine powder.
Do not allow the tissue powder to thaw.
Add liquid nitrogen (enough to cover the tissue) and repeat step10 two more times.
Fig. 1. One gram of leaf sample in mortar with pestle (top-left) and ground with liquid nitrogen (top-right). Home-made mortar holder for liquid nitrogen grinding, made from a Styrofoam block (bottom).
Transfer the ground tissue to a beaker of cold NIB placed on ice with a pre-cooled spatula and mix by gently swirling (Fig. 2). Keep on ice.
On ice
Fig. 2. Ground leaf material in NIB
Repeat the above steps 7 to 12 until all the leaf tissue is ground. The suspension should be smooth with no clumps of leaf material.
Pre-treatment
Pre-treatment
Filtering to remove particulate material. Proceed steps on ice.
On ice
Filter the NIB/sample suspension through 100 μm nylon mesh filter by gravity (see step 5). Do not force the sample through the filter. Replace filters when it is clogged.
On ice
Filter the 100 μm mesh filtrate through the 40 μm nylon mesh (see step 6). Replace filters when it is clogged.
On ice
Divide the filtrate of step 14.2 equally into six Falcon tubes, with 40– 50 ml in each.
On ice
Add 1/20th volume of NIB base-Triton mix, close the tubes with lids and mix the solution by gently swirling and inverting the tubes.
Centrifuge at 2,000 × g for 10 minutes, if possible at 4 °C.
2000 x g, 00:10:00
Carefully discard supernatant without dislodging the pellet.
Add 10 ml Sorbitol buffer to the pellet in each tube and swirl the tubes to mix well.
Centrifuge at 3,000 × g for 10 minutes, if possible at 4 °C.
3000 x g, 00:10:00
Fig. 3. Centrifuged sample after Sorbitol buffer step.
Sample contains a small amount of intact cells (green) and a large amount of nuclei (white).
Discard supernatant leaving the pellet undisturbed. Invert the tubes on tissue to remove traces of buffer without losing the pellet.
Note
The nuclei pellet can now be stored at −80 °C, but this is not recommended.
Note
The pellet might include a small amount of intact cells smaller than 40 μm. Intact cells appear as the greenish in the pellet.
Cell Lysis
Cell Lysis
Set water bath to 50 °C.
50 °C
Add 5 ml of QIAGEN Buffer G2 to each 50 ml Falcon tube containing the nuclei/ cells and gently resuspend the pellet.
Note
The amount of QIAGEN Buffer G2 can be reduced or increased depending on the size of the pellet. Different plant species or tissues may yield different amounts of nuclei/ cells.
Add 0.1% β-mercaptoethanol (5 μl) and a pinch of PVPP to the tube.
Swirl the tube to mix well.
Incubate the tubes at 50 °C for 5 minutes.
50 °C
Add 10 μl RNase A or 50 μl RNase A/T1 Mix to the tube.
Gently swirl the tubes to mix.
Incubate the tubes at 50 °C for 5 minutes for RNase A, or 30 minutes for RNase A/T1 Mix.
50 °C
Add 100 µl QIAGEN Proteinase K to the tube.
Swirl the tubes to mix well.
Incubate the tubes at 50 °C in the water bath, for > 5 hours or overnight.
50 °C
Genomic-tip
Genomic-tip
Set up for QIAGEN Genomic-tip 100/G.
Note
Following the protocol from page 49 of the QIAGEN Genomic DNA Handbook 06/2015.
At this stage in the protocol you will have approx. 30 ml of lysate (six tubes of approx. 5 ml lysate, obtained from 30 g of fresh leaf material ground in 200 ml NIB). It is better not to apply more than 7 ml of lysate to a Genomic-tip 100/G column, since the lysate also contains contaminants and thus an excess amount of contaminants can result in poor DNA quality. Therefore, there are options, to either a) re-using the Genomic-tip column to process multiples of 5 ml lysate from the same sample, or b) use a separate Genomic-tip column for each subsample. For speed and cost savings we use a combination of the two options– two Genomic-tips per sample, reused twice.
Set up Genomic-tip kit: Label 5 × 50 ml Falcon tubes without lids as follows: 1. QBT, 2. Sample, 3. QC 1, 4. QC 2,
5. Final sample.
Note
Use a new Falcon tube for ‘5. Final sample’, the others can be washed, reused tubes.
Note
Optional: Save all the crude extracts and washed-through filtrates and analyze them by electrophoresis to assess the performance of each Genomic-tip step.
Pre-heat Buffer QF to 50 °C in the water bath.
50 °C
To obtain the clear lysate, centrifuge the 50 ml Falcon tubes containing nuclei and cell lysate from step 28 at 3,000 × g for 10 minutes.
3000 x g, 00:10:00
Equilibrating a Genomic-tip 100/G column.
Place the column mounted in the column holder on top of the tube labelled '1. QBT' (see Fig.4).
Fig. 4. Samples applied to a QIAGEN Genomic-tip 100/G column
Add 4 ml Buffer QBT to the column.
Allow the column to empty by gravity flow, do not force the remaining buffer through.
Move the column to the '2. Sample’ tube and apply 5 ml of the clear lysate (supernatant obtained at step 30) to the equilibrated Genomic-tip.
Allow the column to empty by gravity flow.
Wash Genomic-tip 100/G columns with Buffer QC .
Move the column to the ‘3. QC 1’ tube and add 7.5 ml of Buffer QC.
Allow Buffer QC to move through the column by gravity flow.
Move the column to the ‘4. QC 2’ tube and add 7.5 ml Buffer QC.
Allow Buffer QC to move through the column by gravity flow.
Note
Traces of Buffer QC in the column will not affect the elution step.
Eluting the genomic DNA from the Genomic-tip 100/G column.
Move the column to the ‘5. Final Sample’ tube and add 5 ml of Buffer QF pre-warmed to 50 °C.
Allow Buffer QF to flow through the column by gravity.
Store the eluted DNA sample on ice.
Note
The Genomic-tip columns can be reused for the same sample. Simply repeat from step 31– step 34 with the used column. The columns are stable for up to six hours from first equilibration. Do not dry columns.
DNA precipitation & elution
DNA precipitation & elution
Transfer the DNA sample obtained at step 34 to a 2 ml Eppendorf tube.
Add 1 ml sample per tube (totaling 30 tubes from 30 ml elution buffer [5 ml Buffer QF × 6 tubes] at the step 34 in our example).
Add 700 μl of ice-cold isopropanol to each tube and gently invert the tubes to mix.
Leave samples at −20 °C for > 2 hours or overnight.
-20 °C
Centrifuge tubes at 11,000 rpm for 10 minutes.
11000 rpm, 00:10:00
Discard the supernatant.
Note
It is recommended to retain the supernatant until the safe recovery of the DNA pellet has been verified.
Add 400 µl ice cold 70% ethanol to the tubes.
Centrifuge the tubes at 11,000 rpm for 10 minutes.
11000 rpm, 00:10:00
Discard the supernatant by tipping the tube, with the pellet on the upper side, over a clean glass beaker or a Falcon tube.
Note
It is recommended to retain the supernatant until the safe recovery of the DNA pellet has been verified.
Note
Isopropanol pellets have a glassy appearance and may be difficult to see – often they appear (if at all!) as very fine small lines on the side of the tube. To better locate the pellet on the hinge side of the tube, place the tube with the hinge facing outwards in the centrifuge.
Isopropanol pellets are also more loosely attached to the side of the tube, so care should be taken when removing the supernatant. Pure HMW DNA is transparent.
Invert tubes on tissue to remove residual ethanol.
Air dry pellet for approximately 10 – 20 minutes to remove all trace of ethanol.
Note
Do not overdry the pellet as this will make the DNA difficult to dissolve and can also induce nicked-DNA damage.
Add 50 μl TE buffer (or any desired buffer for the downstream application) to the each tube.
Note
If the pellet is not visible, apply the elution buffer to the side of the tube where the pellet is expected to be. Gently flick the tube to rinse the elution buffer over the wall of the tube. Do not pipet or vortex the DNA sample at this stage.
Leave at 50 °C for 1 hour.
50 °C
Note
High molecular weight DNA should be dissolved very gently to avoid shearing.
We incubate the samples at 50 °C in a ThermoMixer with gentle agitation at 300 rpm.
Collect the DNA in two 2ml tubes; i.e., from the 30 tubes with 50 μl DNA sample in each (see step 44), 1.5 ml of DNA sample is obtained, and thus add 750 μl of DNA sample to each tube.
Note
Optional: Check the concentration with NanoDrop to estimate the final concentration. If the concentration is high enough for the downstream application, you can stop here and proceed to the Quality Control steps.
2nd Precipitation to concentrate DNA:
Add 1/10th volume (75 μl) of 3 M NaOAc and an equal volume (825 μl)of ice-cold isopropanol to each tube.
Leave the tubes at −20 °C, for more than 1 hour to overnight.
-20 °C
Centrifuge the tubes at 11,000 rpm for 10 minutes.
11000 rpm, 00:10:00
Discard the supernatant and Invert tubes on tissue to remove residual ethanol.
Air-dry pellet for 10 – 20 minutes. Do not overdry the DNA samples.
Elute the DNA pellet in an appropriate volume of elution buffer depending on the downstream application.
Note
For PacBio genome sequencing, a DNA concentration of > 100 ng/μl is recommended by Kazusa DNA Research Institute (Chiba, Japan). We recommend to add a small volume of elution buffer (e.g. 50 μl) to achieve high concentrations.
Leave the tubes at 50 °C, 300 rpm in a ThermoMixer for 1 hour.
50 °C
Quality Control
Quality Control
Assessing DNA integrity: Perform quality checks for DNA integrity by 0.8% agarose gel electrophoresis or Agilent Technologies TapeStation Genomic DNA ScreenTape.
Assessing DNA quality: Perform a DNA quality assessment for OD values with a spectrophotometer such as NanoDrop or DeNovix.
Assessing DNA quantity: For NGS applications, DNA quantity must be assessed using a fluorometer such as the Qubit or DeNovix.
Note
The DNA concentration values from spectrophotometer and fluorometer assays match in good quality DNA. If they are greatly differ, the DNA quality may be compromised.
Note
Optional: The presence of residual RNA can be measured by Qubit RNA HS assay.