Ultra-long read sequencing for RAD004 with A. thaliana tissue V4
Introduction
This is a modified version of a Josh Quick HMW gDNA prep and ONT sequencing protocol*. It is the fourth version by DRF. It is a branch of V2, not V3 . The major change from V1 to V2 was the use of a CTAB extraction buffer. The major changes from V2 to V4 are the addition of a 2.5M NaCl precipitation at the end of the protocol and the use of Circulomics SRE kit. These additions each increase yield about 10 fold, presumably through the removal of polysaccharides via NaCl and unknown contaminants via SRE. Together, they produce DNA that can yield 3-12 Gbp from a modified RAD-004 MinION run, depending on the MW/quantity of input DNA.
*HMW gDNA prep from Josh Quick of Loman lab, v3 as in https://www.protocols.io/view/ultra-long-read-sequencing-protocol-for-rad004-mrxc57n "Ultra-long read sequencing protocol for RAD004"
Materials
Procedure
Preheat the CTAB buffer to 65C.
Using 1800mg (three densely packed 2ml tubes) of frozen Arabidopsis inflorescence apex, grind tissue in liquid nitrogen to a fine powder using a mortar and pestle.
Keep the ground powder from thawing. Extensive grinding is not needed. Fill a small mortar 3/4 with liquid nitrogen. Add tissue before the liquid nitrogen fully evaporates and lightly grind in the nitrogen. As soon as the nitrogen evaporates grind hard and fast before thawing occurs.
Add 13.44 ml of CTAB buffer directly to mortar, and gently homogenize with pestle. Pour into a 15ml conical tube.
Add 3 ul 100mg/ml RNAse A, and mix by gently inverting 3 times. Place at 37°C for 30 minutes to allow RNase activity.
Add 100 µl Qiagen Proteinase K (20mg/ml, that is 800 units/ml) or other stock to a final concentration of 200 µg/ml. Mix by gently inverting 3 times.
Incubate at 50°C for 1 hour, mix during this incubation at least once by gently inverting 3 times.
50°C for 30 min followed by 65°C for 30 min also worked, unclear if effect on DNA MW
During incubation steps, prepare three 15 ml conical tubes with heavy phase-lock gel if needed.
If gel only available in 2 ml tubes, transfer the gel by cutting the lids off of 2 ml tubes and spinning the gel (1000xg for 1 min) out into 15 ml conical tubes. Four 2ml tubes worth of gel per 15 ml tube.
15 ml conical tubes are used because they are narrower than 50ml, decreasing the surface area of the interface/gel. When mixing, all tubes should be at roughly ~80% of capacity in order to ensure good mixing but limit DNA shearing.
Spin at 3600 xg for 6 minutes to precipitate solid matter.
Pour 6 ml of the supernatant into each of two 15 ml conical tubes prepared with phase-lock gel.
Add 7 ml PCI to each 15 ml tube.
Place on a RotoMini plus (rotisserie mixer) at 10 rpm for 10 minutes with the tube horizontal to the plane of rotation at a roughly 5 degree angle off from the plane.
Watch to ensure that an emulsion is formed but mixing should not be violent.
Spin in a centrifuge at 4120 xg for 10 minutes.
Combine the two aqueous phases by pouring into the last 15 ml conical tube containing phase-lock gel. Typically this recovers about 10.5 ml with the gel.
Add 3.3 ml (1/3X volume) of chloroform to the 15ml tube.
Place on a RotoMini plus (rotisserie mixer) at 10 rpm for 10 minutes with the tube horizontal to the plane of rotation at a roughly 10 degree angle off from the plane.
Spin in a centrifuge at 4120x g for 10 minutes.
Pour the aqueous phase slowly into a new 50 ml Falcon tube.
!!! The phase lock gel may be very loose after the chloroform extraction. Pour carefully but not too slowly. Do not invert the pouring tube more than needed. Alternatively use a wide-bore pipette to transfer.
Add 4.5 ml of 5M NaCl to 10.5 ml of aqueous phase (to bring to 2.5M NaCl) and mix by inverting gently twice.
Add 14.5ml (one volume) of 4C isopropanol, invert gently three times and place at 4C for 30 min.
Bubble should visibly form, a weak phase separation of some kind should form, and a cobweb of DNA should be visible by the end of this step.
Use a glass hook to fish out the DNA (in one-piece). Slowly swirl the hook to wind the DNA onto it. Allow the excess liquid to drip off. Immediately submerge the DNA in a 5 ml Eppendorf tube containing 5ml of fresh 70% ethanol. It should tighten up on the hook slightly.
You may need to spin the 50ml tube at 300xg for 1 minute to get the larger DNA into a loose mass at the bottom before fishing it out. Recovery of a second fraction of lower MW DNA, may be achieved by a second longer/harder spin.
Make a hook by melting the tip of glass capillary in a blue flame so it curls over.
Carefully work the pellet off the hook, using the rim of a microfuge tube or a pipette tip. Let the pellet drop into the microfuge tube.
Add 500 ul of 70% ethanol to the microfuge. Resist the temptation to mix.
Spin down at 10,000 x g then remove as much of the 70% ethanol as possible.
Let the remaining ethanol evaporate by leaving at RT for 30 seconds.
Add 90ul EB and incubate at 4°C for 2 days in the dark to allow the pellet to resuspend into a translucent viscous gel.
Check your yield by quantifying the DNA using the Qubit BR assay. It is important to use BR as the buffer contains a detergent which appears to improve mixing. Yield for 1g of tissue ought to be roughly 25ug of DNA but qubit readings may vary substantially because of the non-homogeneity of the solution.
I typically quantify using two qubit reactions with 1ul of DNA prep in each since the DNA prep can be very non-homogenous. After adding the DNA prep to the qubit mix, vortex at full speed for 30 seconds and let sit for 10 minutes as the input DNA may still need to fully resuspend after addition to qubit mix.
Place the entire DNA prep through the SRE kit, diluting as necessary so as not to exceed the recommended 150ng/ul. Resuspend in 20 ul EB.
20 ul EB resuspension is used in order to have 1 ul for Qubit and 18.5 ul for the RAD-004 run.
Resuspending in EB+Triton-X100 supposedly improves the transposase activity.
Place resuspending DNA at 37C for 1 hour if performing sequencing same day. Otherwise, 4C overnight.
Quantify with Qubit BR using 1 ul of SRE DNA. Optionally, perform a CHEF gel and/or Nanodrop.*
Prepare for library preparation
- Allow flowcell to warm at RT for 5 minutes, then perform flowcell check.
- Preheat the library prep program on the thermal cycler - 30°C for 1 min, 80°C for 1 min, hold at 22°C
- Thaw the reagents at RT - LB, SQB, FLT, FB. Do not vortex FLT.
As slowly as you can pipette, move 18.5 µl DNA solution into a PCR tube using a wide-bore P20 pipette tip. Retain the tip.
Add 1.5 µl FRA. Using the same tip from the last step, mix up and down as slowly as possible 7 times. Very briefly spin.
If Triton has been added, take care not to introduce bubbles as they are hard to remove.
Place the tube in the thermal cycler and start the program.
Immediately when done, add 1 µl RAP. Using a P20 set to 19 µl with a wide-bore tip, mix up and down as slowly as possible 7 times.
Incubate at room temperature for 5 minutes. Proceed to prime the flowcell while waiting. Place the library on ice after 5 minutes if you have not finished priming.
Add 30 µl FLT to a tube of FLB and mix by inverted several times. This is the priming mix.
Open the priming port. Using a P1000, remove a 20-30ul storage buffer from the priming port using the pipette's volume adjustment screw.
Load 800 µl priming mix via the priming port. Leave the port open.
Initially you will need to slowly pipette by spinning down the volume adjustment to start the priming. After a small initial volume goes in, the rest can be dripped onto the priming port.
Wait for at least 5 minutes before performing the second priming. Proceed to mixing the library while waiting.
Prepare the final library by adding 34 ul of SQB and 20 ul of the supernatant of spun-down LB. Using a P200 set to 70 µl with a wide-bore tip, mix up and down as slowly as possible 4 times.
Open the SpotON port, then load 200 µl flush mix into the priming port dropwise. Half way through the 200ul, place one drop onto the SpotON port itself. Continue to leave the ports open.
Using a P200 set to 70 µl with a wide-bore tip, mix up and down as slowly as possible 3 times. On the final mix, slowly pipette 'spin' the final library onto the SpotON port dropwise.
The library should get siphoned in, but this can take much longer than usual due to the viscosity. If it gets blocked, load by holding the pipette vertically against the SpotON port and pipette 'spinning' the library directly in.
Close the priming port then close the SpotON port.
Start the run via MinKNOW using default settings for RAD-004. Time between mux may be increased if expecting extraordinarily long outputs.
Run a CHEF gel if testing DNA lengths. 500ng DNA in a lane, 1ug preferred.
Pour 1% gel with Certified Megabase Agarose in TAE. S. cerevisiae and lambda are useful ladders.
Run at 160V (5V/cm) set for 22 hours from 50s to 90s volt time ramp in cold room with prechilled TAE buffer. Buffer pump set to 50.
End run between 18 and 22 hours after start.
Check the absorbance spectrum of 1ul of 1:2 diluted DNA on the NanoDrop.
The Nanodrop typically drastically overestimates the quantity of DNA.
An example prep prior to SRE: 260/80=1.98 and 260/230=2.29
