TITLE: From low cost plant HMW DNA extraction to MinION sequencing
AUTHORS: Julien Serret, marie.couderc , Cedric Mariac, Laurencealbar , Francois Sabot

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This low cost protocol is efficient to extract high molecular weight DNA from several tropical plants to prepare LSK109 libraries for Nanopore sequencing. Pulsed-field gel electrophoresis reveals a DNA size range from 40 to 350 Kb for four species of rice, Paspalum, date palm and </span><span style = "font-style:italic;">Ocotea obtusata</span><span>. Sequencing on MinION Nanopore flow cells yielded from 3.3 to 25.3 Gb with reads N50 values ranging from 15.4 to 31.3 Kb.</span></div></div>

[STEPS]
?. [Plant materiel sampling]
Collect up to  of fresh young leaves in a 50ml clean tube.Grind sample with liquid nitrogen, mortar and pestel to a fine powder.An analytical grinder (A15, IKA, Germany) can also be used for 2x  .
5 g
?. [Tissue lysis]
Add  of freshly prepared MATAB lysis buffer (100mM Tris pH8, 1.4M NaCl, 20mM EDTA ph8, 1% PEG6000, 2% MATAB, 1mM DTT).Homogenize slowly by inversion.Incubate at  for  and mix by gentle inversion every  .Add  of proteinase K (20 mg/ml; eg from Terra PCR Kit) and incubate at   for  with homogeneisation by inversion every  .
15 mL
65 °C
2.5 µl
55 °C
?. [DNA purification]
Add  of  and homogenize slowly  manually or with horizontal shaker   .Centrifuge  and carefully transfer the supernatant to a clean 50 ml tube by using a 1000µl pipette tip cut at the extremity.Add  of  , slowly homogenize and incubate  at  .Add  of  and agitate slowly manually or with horizontal shaker  .Centrifuge  and carefully transfer the supernatant to a clean 50 ml tube by using a 1000µl pipette tip cut at the extremity..
15 mL
33
Centrifuge: 4000 33, 20 min, 20 10
Optionnal:If the supernatant is not clear, add   of   and agitate slowly manually or with horizontal shaker   .Centrifuge   and carefully transfer the supernatant to a clean 50 ml tube by using a 1000µl pipette tip cut at the extremity..
16 µl
0 Room temperature
13 mL
15 33
Centrifuge: 4000 33, 20 min, 20 10
?. [DNA precipitation]
Add   of 3M Sodium acetate pH5.2 and   of .Homogenize by very slowly inverting the tube until DNA precipitate appears.Glass hook DNA and transfer into a 2 ml Eppendorf tube containing 1 ml 70% EtOH.Then, air dry the DNA precipitate  .Transfer the DNA precipitate into  of ultrapure water in a 1.5ml Eppendorf tube.Let the DNA resuspend  at
300 µl
4 °C
?. [DNA clean-up]
Resuspend the equilibrated at room temperature by vortexing.In a 2ml Eppendorf tube, add  of    to  of DNA.Homogenize by gently flicking the tube and incubate   at  .Spin down and pellet on the magnet.Carrefully pipette off the supernatant and wash twice with  of fresh 70% ethanol.Remove the tube from the magnet and add  of ultrapure water.Incubate at   until the beads are completely resuspended (approximately 2x ). Help resuspension by gently flicking the tube.
To avoid DNA shearing, always cut tip extremities using a clean razor blade before pipetting.
150 µl
150 µl
0 Room temperature
500 µl
150 µl
65 °C
?. [DNA quantification]
Quantify 1µl of DNA using a Qubit fluorometer.
?. [Check HMW using Pulsed-Field Gel Electrophoresis]
Molecular weight of extracted DNA was assessed by PFGE by loading   of DNA ( to   of DNA,  of 6x loading buffer) and into 1% agarose gel and using the following parameters for migration:Pulse time : initial = 5, final = 120Run time = 18 h V/cm = 5 Angle = 120 Temp = 14° mA end of run = 255
20 µl
1.5 µg
5.5 µg
5 µl
?. [Short read elimination]
Perform short read elimination with a 25Kb cut-off by following the Circulomics SRE kit (SKUSS-100-101-01) instructions.Incubate  at  in elution buffer before Qubit quantification and library preparation.
4 °C
?. [SQK-LSK109 library preparation]
SQK-LSK109  library preparation is performed according to  protocol with several modifications :
?. [SQK-LSK109 library preparation]
DNA repair & end-prepIn a 1.5ml Eppendorf DNA LoBind tube mix: DNA sample (9µg of extracted DNA with volume adjusted with 50/50 Ultrapure water/TE pH8) NEBNext FFPE DNA Repair Buffer Ultra II End-prep Reaction Buffer  Ultra II End-prep Enzyme MixMix by gently flicking the tube, and spin down.Using a thermal cycler, incubate at  for  and   for  .Resuspend the equilibrated at room temperature by vortexing.Add  of  to the end-prep reaction and mix by gently flicking the tube.Incubate  at  .Meanwhile, prepare  of 70% ethanol using nuclease-free water.After the incubation, spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.Wash the beads twice with  of freshly-prepared 70% ethanol.Spin down and place the tube back on the magnet to pipette off the residual ethanol.Remove the tube from the magnet and resuspend the pellet in  nuclease-free water.Incubate  at  and  at  . Gently flick the tube and incubate  more at  .Pellet the beads on a magnet until the eluate is clear and colourless.Remove and retain  of eluate into a clean 1.5ml Eppendorf DNA LoBind tube.Quantify  of eluted sample using a Qubit fluorometer.
47 µl
3.5 µl
2 µl
3.5 µl
3 µl
20 °C
65 °C
50 µl
0 Room temperature
500 µl
200 µl
62 µl
0 Room temperature
65 °C
65 °C
62 µl
1 µl
?. [SQK-LSK109 library preparation]
Adapter ligationIn a 1.5ml Eppendorf DNA LoBind tube, mix: DNA sample from the previous step Ligation Buffer (LNB) NEBNext Quick T4 DNA Ligase Adapter Mix (AMX)Mix by gently flicking the tube, and spin down.Incubate the reaction for  at Resuspend the  equilibrated by vortexing.Add  of  to the end-prep reaction and mix by gently flicking the tube.Incubate  at  .Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.Wash the beads by adding  of Long Fragment Buffer (LFB) at room temperature. Remove the supernatant and repeat the wash step.Spin down and place the tube back on the magnet to pipette off the residual LFB.Remove the tube from the magnet and resuspend the pellet in  of Elution Buffer (EB).Incubate  at  and   at .Pellet the beads on a magnet until the eluate is clear and colourless.Remove and retain  of eluate into a clean 0.5ml Eppendorf DNA LoBind tube.Quantify  of eluted sample using a Qubit fluorometer.
61 µl
25 µl
10 µl
5 µl
20 °C
0 Room temperature
50 µl
0 Room temperature
250 µl
16 µl
37 Room temperature
37 °C
16 µl
1 µl
?. [MinION sequencing]
Split the 16µl library into two 8µl in 0.5ml Eppendorf DNA LoBind tube. Store one tube at  until use.
4 °C
?. [MinION sequencing]
Day 1 - First Load In the second tube, add to the 8µl library: Elution buffer (EB) Sequencing buffer (SQB) Loading beads (LB), mixed immediately before use.Using MinION mk1C device, perform the flow cell check. (FLO-MIN106D - Spot-ON Flow Cell R9 version)Prepare the priming mix by adding   of Flush tether (FLT) directly into a fresh  Flush buffer (FB) tube.Mix by vortexing.After opening the priming port, check for a small air bubble under the cover. Draw back a small volume toremove any bubbles (a few μl):- Set a P1000 pipette to 200 μl- Insert the tip into the priming port- Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering thepipette tip.Load  of the priming mix into the flow cell via the priming port, avoiding the introduction of airbubbles. Wait for .Remove again any bubbles : - Set a P1000 pipette to 200 μl- Insert the tip into the priming port- Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.Gently open the SpotON sample port.Load an additional 200 µl of the priming mix into the still open priming port.Mix the prepared library gently by pipetting up and down just prior to loading.Slowly, add  of the library into the SpotON sample port. Make sure the priming port is still open during this step and the library should easily fall into the sample port.Close the priming port and replace the SpotON sample port cover.Close the lid of the MinION device.Start the sequencing experiment by setting a 96h run.
4 µl
37.5 µl
25.5 µl
30 µl
800 µl
75 µl
?. [MinION sequencing]
Day 3 - Second Load After 48h running, pause the sequencing run.Perform the flow cell washing step according to the ONT protocol (EXP-WSH003).At the end of this protocol, re-prime the flow cell as indicated in the 10.1 step.In the 8µl library tube stored at 4°C, add the following: Elution buffer (EB) Sequencing buffer (SQB) Loading beads (LB), mixed immediately before use.Load on the flow cell as the 10.1 step.Resume the sequencing run.
The wash kit allows to revert "unavailable pores" into "active pores".This procedure provides the opportunity to maximise the flow cell yield.
4 µl
37.5 µl
25.5 µl
?. [MinION sequencing]
After 72h running, if translocation speed falls below 250 bases per seconde, pause the run and refuel:After opening the priming port, check for a small air bubble under the cover. Draw back a small volume toremove any bubbles (a few μl):- Set a P1000 pipette to 200 μl- Insert the tip into the priming port- Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.Load   of Flush Buffer (FB). Close the priming port and resume the run.
?. [Results]