TITLE: FindingNemo in OneDay: Ultra-Long ONT Library Preparation from Cell to Flowcell in One Day
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is focussed on the isolation of ultra-high molecular weight (UHMW) DNA, library preparation and clean up ready for sequencing on the Oxford Nanopore Technology (ONT) platforms, all within </span><span style = "font-weight:bold;">one working day</span><span>. </span></div><div class = "text-block">This protocol is optimised for human cell lines. </div><br/><div class = "text-block"><span style = "font-weight:bold;">Summary of the workflow:</span></div><div class = "text-block"><div class = "justify" style = "text-align:center"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;"> </span></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/ecn69wh6.jpg" /></div><br/></div>

[STEPS]
?. [Monarch UHMW DNA Extraction Kit]
We have obtained optimal DNA extractions using the NEB Monarch kit. This combines speed with high quality UHMW DNA. Follow the manufacturer’s instructions as described here, BUT incorporate the following changes as described below.
Our most homogeneous extracted DNA samples were obtained by lysis at 600-700 rpm speed using the Monarch Kit. This is one area that can be optimised depending on the input sample.
?. [UHMW DNA QC]
Two nucleic acid quantification methods, i.e., fluorometric (Qubit) and spectrophotometric (Nanodrop), can be used in parallel to assess both the quantity and purity of the extracted DNA. The quantification follows the published protocol by Koetsier and Cantor with slight modifications as follows.An accurate measurement of DNA concentration is important as this will determine the optimum ratio of transposase to DNA molecules at the library prep step. Also, the viscous nature of UHMW DNA requires that sample measurement represents all parts of the DNA solution.A total of 10 μl DNA is sampled from four different locations in the tube: topupper-middlelower-middlebottomEach sample should be 2.5 μl and combined into a single 2 ml tube.
Use a cut P10 tip to aspirate the DNA sample and, if too viscous, cut the DNA thread by pushing the tip against the bottom of the tube. When available, a positive displacement pipette can also be used to ensure more accurate liquid aspiration.
?. [UHMW DNA QC]
Add a glass bead and pulse vortex at full speed for one minute.
33
?. [UHMW DNA QC]
Quantify and calculate %CV as described in this paper by Koetsier and Cantor.
Typically we take three measurements of 1 μl each on Nanodrop and one measurement of 3 μl using the Qubit DNA BR kit (standardized by the Jurkat genomic DNA).
?. [UHMW DNA QC]
Next quantify any RNA carry-over using the Qubit RNA Broad Range kit (optional).
This step is to confirm that the RNA content is low to ensure maximum amount of DNA. It should be less than 50% of the DNA concentration measured with Qubit.
?. [UHMW DNA QC]
Wherever possible, the quality of extracted DNA sample should be analysed by method(s) that enable visual inspection of molecule length distribution such as:Regular agarose gel electrophoresisPulsed-Field Gel Electrophoresis, e.g., using Pippin Pulse (Sage Science)Agilent Bioanalyzer DNAAgilent TapeStation DNA
?. [UL Library Prep]
For library preparation, follow the "Modified ULK001" library prep protocol (modified from Oxford Nanopore Technologies SQK-ULK001) as linked below:
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?. [NEMO Library Clean-up]
This section provides an alcohol-free purification of a nanopore DNA sequencing library from an UL protocol.For 5-40 μg of input UHMW DNA (corresponding to DNA extracted from 1-6 million human cells), add 3 clean glass beads into the sample in a 2 ml tube.
For DNA amounts less than 5 μg, 2 glass beads can be used (see table at step 23). Two slightly larger glass beads from New England Biolabs (NEB; see Materials) can also be used per reaction. Glass beads can be washed following an acid-, bleach-, or SDS-wash protocol then sterilized. Sterilization can be by autoclaving or just storing the beads in 70% Ethanol.
?. [NEMO Library Clean-up]
Add a 1:1 volume of 10 mM Hexamminecobalt(III) Chloride (CoHex) into the DNA solution.
When the clean-up follows ONT ultra-long library preparation (SQK-ULK001), volumes can typically range from 500-1000 μl.
?. [NEMO Library Clean-up]
Rotate the tube with a vertical rotator at 9 rpm for 5-10 minutes.
33
[10m max]
Rotate for 5 minutes if the DNA amount is less than 5 μg and adjust the time when more DNA is used, up to 10 minutes for 40 μg DNA. If a rotator is not available, hand inversion for 30-40 repeats can be used. Invert the tube slowly by hand such that each full cycle takes around 5 seconds.
?. [NEMO Library Clean-up]
Invert the tube 3 times more by hand to ensure DNA has precipitated and is tightly bound to the beads.
?. [NEMO Library Clean-up]
Discard the supernatant. Take care not to disturb the DNA precipitated onto the beads.
?. [NEMO Library Clean-up]
Wash the glass beads by gently adding 1 ml of PEGW buffer and gently invert 3 times. Incubate for 3 minutes at room temperature.
0 Room temperature
?. [NEMO Library Clean-up]
Discard most of the supernatant, again taking care not to disturb the DNA precipitate.
?. [NEMO Library Clean-up]
Repeat step 17 with 500 μl of the PEGW buffer.
?. [NEMO Library Clean-up]
Discard the supernatant, taking care not to disturb the DNA precipitate. It isn't necessary to remove everything, a small volume of liquid can be left behind.
?. [NEMO Library Clean-up]
Insert a bead retainer into a collection tube.
?. [NEMO Library Clean-up]
Pour the beads from step 20 into the bead retainer and pulse-spin for 1 second in a mini centrifuge (or the shortest time possible) to remove any residual wash buffer. Keep the bead retainer.
Omitting Dry-spin StepWhen working with large amounts of DNA, this dry-spin step can be omitted to prevent DNA loss from spinning. Follow steps below:Remove the supernatant after the second wash as much as possible, pulse-spin the tube for 1 second and remove last traces of buffer from under the glass beads with a fine pipette tip. Proceed to the elution step.
?. [NEMO Library Clean-up]
Quickly pour the beads into a new 2 ml low-bind tube and immediately add the corresponding volume of elution buffer  (ONT-EB or 10 mM Tris-HCl pH 8.0) per the table below.DNA Input Amount (μg)No. of glass beads 
(3-mm diameter)Elution Buffer Volume (μl)>30-403225>20-303180>5-203120>2-52901-2150
DNA Input Amount (μg)No. of glass beads 
(3-mm diameter)Elution Buffer Volume (μl)>30-403225>20-303180>5-203120>2-52901-2150
Do not let the beads with DNA dry out, as it will make DNA homogenization into solution more difficult. As an alternative, the elution buffer can be aliquoted into a 2 ml tube prior to this step. The beads can then just be poured into the buffer.
?. [NEMO Library Clean-up]
Incubate the library at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P200 tip to aid elution.
37 °C
[mix per 10 min]
?. [NEMO Library Clean-up]
Insert the bead retainer from step 22 into a clean 1.5 ml tube. Pour the beads from step 24 into the bead retainer and centrifuge at 12,000 x g for one minute.
Centrifuge: 12000 33, 1 min
?. [NEMO Library Clean-up]
Incubate for at least 30 minutes at room temperature with regular pipette mixing.
0 Room temperature
[at least]
?. [NEMO Library Clean-up]
Now - you have found Nemo!       Store the library at 4oC or continue loading it to a flowcell.
[for storage]
Load at least 1 μg for MinION sequencing (or all of the library for input cells of 1 million).
?. [Flowcell Priming & Library Loading]
Prime the flow cell as per the MinION or PromethION protocol.YouTube Video: Priming A MinION Flowcell
?. [Flowcell Priming & Library Loading]
Quantify 2-3 μl of the library sample using fluorometric method (Qubit DNA BR kit) or alternatively the spectophotometric method (Nanodrop).
?. [Flowcell Priming & Library Loading]
Mix 38-40 μl library (or at least 1 μg) with the same volume of sequencing buffer (SQB) from the SQK-ULK001 kit or SQK-RAD004, mix and incubate at room temperature for 30 minutes.
0 Room temperature
?. [Flowcell Priming & Library Loading]
Load the library per SQK-ULK001 protocol and let it tether for another 30 minutes before starting the run.
[tethering]
?. [Flowcell Priming & Library Loading]
Select the correct UL sequencing script based on the sequencing kit used (default mux scan should already be set to every 6 hours).
?. [Home-brew Flowcell Wash/Flush (Optional)]
This section can be used to reload library on the same flowcell.Add 2 μl DNase I to 398 μl nuclease flush buffer (NFB), vortex to mix.
?. [Home-brew Flowcell Wash/Flush (Optional)]
After opening the priming port of the flow cell, check for a small bubble under the cover. Draw back a small volume to remove any bubble:
?. [Home-brew Flowcell Wash/Flush (Optional)]
Set a P1000 pipette to 200 μl
?. [Home-brew Flowcell Wash/Flush (Optional)]
Insert the tip into the priming port
?. [Home-brew Flowcell Wash/Flush (Optional)]
Turn the wheel until the dial shows 220-230 μl, or until a small volume of buffer is seen entering the pipette tip
?. [Home-brew Flowcell Wash/Flush (Optional)]
Using a P1000 pipette, load 400 μl of the NFB plus DNase I into the flow cell priming port.
?. [Home-brew Flowcell Wash/Flush (Optional)]
Close the flow cell priming port and incubate the flow cell in situ for at least 1 hour.
[at least]
?. [Home-brew Flowcell Wash/Flush (Optional)]
Reprime the flow cell as in step 28.
?. [Home-brew Flowcell Wash/Flush (Optional)]
Reload the library as in step 30-32..
?. [Monarch UHMW DNA Extraction Kit]
To complete in one day, start early!
One million human cells are sufficient for a single library load on the MinION. At least two million human cells are required for a single load on the PromethION. Other samples can be scaled according to total amount of DNA recovered. So for a 1 gigabase genome you would require 3 million cells etc.
?. [Monarch UHMW DNA Extraction Kit]
Incubate the eluted DNA at 37oC for 2-3 hours with regular pipette mixing.
37 °C
[max (2-3 hours)]
During mixing, observe by eye that the viscous DNA 'blob' has been more or less dissolved to the different parts of the tube (i.e., less heterogeneous). This is usually observed after 2 hours of incubation. Otherwise, continue the incubation to 3 hours and proceed to the next step.
?. [Monarch UHMW DNA Extraction Kit]
Quantify the DNA as per "UHMW DNA QC" (steps 6-10 below) and check homogeneity by calculating %CV values. If the DNA is not sufficiently homogeneous (i.e., %CV ≥ 100%) incubate the DNA for longer.
?. [Monarch UHMW DNA Extraction Kit]
Dilute the eluted DNA with 150 μl of NEB Elution Buffer II.
This is following the NEB UHMW Monarch kit protocol where DNA is first eluted with 100 μl elution buffer. After this step, sample volume will be 250 μl total. Quantification of very viscous UHMW DNA is problematic and will not produce accurate results, hence the dilution. Gradual dilution is recommended to achieve a homogeneous final concentration of 50-100 ng/μl.