1. Prepare for your experiment
Extract DNA from the samples with the right kits, and check the following:
- Length (Bioanalyzer/ Tape Station)
- Quantity (Level of degradation)
- Purity (no protein contamination)
The quality controls performed during the protocol are essential.
2. Shear the DNA with g-Tube
1) Transfer ~1 μg purified DNA to Eppendorf DNA LoBind tube and adjust the volume to 49 ul with TE buffer.
2) Mix well and transfer the DNA sample to a Covaris g-TUBE.
3) Centrifuge the g-TUBE for 4min at R.T., at 2500 xg with Eppendorf 5424 microfuge.
4) Revert the the g-TUBE and centrifuge again under same condition.
5) Transfer the ~ 49 ul fragmented DNA to a clean 1.5ml Eppendorf DNA LoBind tube.
3. End-prep and DNA repair
1) In a 0.2 ml 96 well PCR plate, set up the reactions:
Fragmented DNA 48 ul
Ultra II End-prep reaction buffer 3.5 ul
Ultra II End-prep enzyme mix 3 ul
FFPE Repair buffer 3.5 ul
FFPE Repair Mix 2 ul
2) Mix by pipetting. Spin down in a centrifuge and incubate for 30 minutes at 20 °C and 30 minutes at 65 °C using the thermal cycler. Set the lid heat to 75°C.
3) Resuspend the room temperature AMPure XP beads by vortexing. Add 60 μl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.
4) Let DNA bind to beads for 5 minutes at room temperature, using a rotator.
5) Place the tube on magnetic rack, and pipette off supernatant when it’s clear.
6) Keep the tube on the magnet and wash the beads with 180 μl of freshly-prepared 80% ethanol without disturbing the pellet. Discard the ethanol using a pipette.
7) Repeat the previous step.
8) Air the dry the beads for ~2min. Do not dry for too long. [good when matte, not glassy, no cracks]
9) Remove the tube from the magnet and resuspend pellet in 31 μl nuclease-free water. Incubate for 5 minutes at room temperature.
10) Pellet the beads on a magnet until the eluate is clear.
11) Remove eluate once it is clear. Transfer the eluted sample to a new 0.2 ml PCR tube.
12) Quantify 1 μl of end-prepped DNA using a Qubit fluorometer - recovery aim > 700 ng.
4. Ligation of Barcode Adapter
1) Add the reagents to a fresh 0.2ml PCR tube:
End-preped DNA. 30 ul
Barcode Adapter (blue cap) 20 ul
Blunt/TA Ligase Master Mix. 50 ul
2) Mix by pipetting. Briefly spin and incubate the reaction for 30 minutes at room temperature.
3) Resuspend the AMPure XP beads by vortexing. Add 45 μl of resuspended AMPure XP beads to each sample and mix by pipetting up and down ten times.
4) Let the DNA bind to beads for 5 minutes at room temperature.
5) Place the tube on magnetic rack, and pipette off the supernatant when it’s clear.
6) Keep the tube on the magnet and wash the beads with 180 μl of freshly-prepared 80% ethanol without disturbing the pellet. Discard the ethanol using a pipette.
7) Repeat the previous step.
8) Air the dry the beads for ~2min. Do not dry for too long. [good when matte, not glassy, no cracks]
9) Remove the tube from the magnet and resuspend pellet in 30 μl nuclease-free water. Incubate for 5minutes at room temperature.
10) Pellet the beads on a magnet until the eluate is clear. Transfer the eluted sample to a new PCR tube.
5. RE-treatment for samples
1) Add the reagents to a fresh 0.2ml PCR tube, in the order given below:
10X Tango Buffer 5 ul
Adapter-ligated DNA 27 ul
NF water 16 ul
XapI Restriction Enzyme 2 ul
2) Mix well and briefly spin down. Incubate the RE reaction for 30min at 37°C.
For different RE, check the manufacturer’s instruction for temperature and buffer.
3) Terminate the reaction by adding 10ul loading dye (6X) to the reaction.
6. Gel purification
1) Set up the agarose gel
-Weigh 0.6g of agarose and dissolve with 65 ml 1XTAE buffer.
-Cool down the gel buffer to 80°C and add 6ul Gel Red dye to the dissolved gel buffer.
-Pour the gel in the gel cassette
-Loading the DNA in to the well. Load 12ul DNA ladder. Run the gel at 140v voltage in 1X TAE buffer till the loading dye reached 2/3 of the gel
2) Visualize the gel with Image lab. Then cut out the band (5kb-10kb, indicated by DNA marker) with the UV transilluminator. Transfer the gel to a new 1.5 ml Eppendorf tube, the weigh the gel with a balance.
3) Extract the gel with the Gel and PCR Clean-up Kit (Macherey-Nagel, 740609.250)
4) Add 200ul NTI/ 100mg gel.
5) Incubate the sample at 50°C for 10min. Vortex the sample briefly every 2-3 min till the gel slice is completely dissolved!
6) Load 700 ul sample to the NucleoSpin Gel and PCR Cleanup Column. Centrifuge for 30s at 11,000 x g. Discard the flow-through. Load remaining sample if necessary and repeat the centrifuge step.
7) Wash the silica membrane with 700ul Buffer NT3. Centrifuge for 30s at 11,000 x g. Discard the flow-through. Repeat the wash step.
8) Centrifuge the NucleoSpin Gel and PCR Cleanup Column at 11,000 x g for 1 min to remove the Buffer NT3 completely.
9) Elute the DNA by adding 20ul of NF-water to the silica membrane. Incubate at R.T. for 1 min and centrifuge for 1min at 11,000 x g.
7. Sample Barcoding PCR
1) Set up a barcoding PCR reaction as follows for each sample:
PCR Barcode (one of BC1-BC96, at 10 μM) 1 ul
Gel purified DNA 24 ul
PrimeSTAR GXL 2x Master mix 25 ul
2) Mix well. Briefly spin down.
3) Amplify using the following cycling conditions:
98°C for 1 min followed by 15-18 cycles of 98°C for 15 s, annealing 62°C for 15 s and extension at 68°C for 8 min, with a final extension at 68°C for 10 min.
4) QC step
- Qubit. Use 1ul of PCR product for Qubit, the concentration for an efficient PCR is ranging 10-50 ng/ul. If the concentration is low, add 2-4 more cycles.
- Gel. Use another 2ul for a gel picture to check the PCR product size, correct one is around 5-10kb.
5) Purify the barcoded DNA using standard methods which are suitable for the fragment size. Add 50ul NF water to the PCR product and then add 50 ul Ampure XP beads (0.5X beads).
6) Quantify the barcoded library using standard techniques, and pool all barcoded libraries in the desired ratios in a 1.5 ml DNA LoBind Eppendorf tube.
8. Library Preparation
1) Sample pooling
-IMPORTANT: This is the guidance for sequencing with a MinION R9.4.1 flow cell.
a. Total amount for pooled library: 1000 ng
b. Transfer each barcoded library with equal amount to a new tube. Adjust the volume to 47 ul with NF water.
- If the pooled library volume is more than 47 ul, concentrate the sample with 2X beads and then elute with 47 ul NF water.
2) DNA repair and end-prep
a. Thaw the DNA CS (DCS) at room temperature, spin down, mix by pipetting, and place on ice.
b. Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
c. In a 0.2 ml thin-walled PCR tube, mix the following:
Pooled DNA library 47ul
DNA CS 1 ul
Ultra II End-prep reaction buffer 3.5 ul
Ultra II End-prep enzyme mix 3 ul
FFPE Repair buffer 3.5 ul
FFPE Repair Mix 2 ul
d. Mix gently by flicking the tube, and spin down.
e. Incubate at 20°C for 15 minutes and 65°C for 15 minutes on a thermal cycler
!!! Set the lid heat to 75°C.
f. Add 60 μl of resuspended AMPure XP beads to the end-prep reaction and mix by flicking the tube. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
g. Spin down the sample and pellet on a magnet until eluate is clear. Pipette off the supernatant without disturb the beads.
h. Wash the beads with 200ul freshly prepared 80% EtOH for twice.
i. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
j. Resuspend the pellet in 31 μl nuclease-free water. Incubate for 5 minutes at room temperature.
k. Transfer 31 μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
3) Adapter ligation and clean-up
a. Preparation:
- Spin down the Adapter Mix (AMX) and Quick T4 Ligase, and place on ice.
- Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
- Thaw the Elution Buffer (EB) at room temperature, mix by vortexing, spin down and place on ice.
- Depending on the wash buffer (LFB or SFB) used:
o To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.
o To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, spin down and place on ice.
b. In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:
c. Mix gently by tapping/ flicking the tube, and spin down. Incubate the reaction for 30 minutes at room temperature.
d. Add 40 μl of resuspended AMPure XP beads to the reaction and mix by flicking the tube. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
e. Spin down the sample and pellet on a magnet until eluate is clear. Pipette off the supernatant without disturb the beads.
f. Wash the beads with either 125 μl Long Fragment Buffer (LFB) or 125 μl Short Fragment Buffer (SFB) for twice. Resuspend the beads by flicking, spin down, then remove the supernatant and discard.
g. Remove any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
h. Resuspend the pellet with 7 μl Elution Buffer (EB) and incubate for 10 minutes at room temperature. For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.
i. Transfer 7 μl of eluatedDNA library into a clean 1.5 ml Eppendorf DNA LoBind tube, then take 1 ul for quantification using a Qubit fluorometer.
9. Sequencing with ONT
1) Preparation:
a. Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing.
b. Prepare the priming mix by adding 30 ul of the thawed FLT to 1170 ul of FB, mix well.
c. Insert the flow cell by slide the flow cell under the clip of the MinION device.
2) Priming and loading Flow Cell
a. Open the priming port by sliding the flow cell port cover clockwise.
b. Draw back a small volume to remove any bubbles using a P1000 pipette.
c. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Incubate for 5 minutes.
d. Prepare the library loading mix in a new tube during the 5min incubation:
SQB 37.5 ul
LB, mixed immediately before use 25.5 ul
DNA library 12 ul
e. Lift the SpotON sample port cover, then complete the flow cell priming by adding 200 ul of the priming mix into the flow cell priming port.
f. Mix the library loading mix well by gentle pipetting.
g. Add the 75 ul of the library loading mix trhough the SpotON sample port in a dropwise way. Enure each drop flows into the port before adding the next drop.
h. Gently cover of the SpotON sample port and slide off the priming port.
3) Data collection
Data acquisition is carried out by the MinKNOW software.