FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms

A DNA extraction protocol that yields clean and homogeneous UHMW DNA is essential for good UL sequencing output. The choice of protocol should be based on achieving these parameters.

We provide alternatives to achieve this by following kit-based or kit-free extraction protocols as follows:

1. Kit-free, phenol-based : a scaled-down version of Josh Quick's protocol (dx.doi.org/10.17504/protocols.io.mrxc57n) with additional glass bead step for DNA precipitation.
2. Kit-free, phenol-free : a modification of NEB's Monarch HMW DNA Extraction Kit for Cells & Blood, with the option to use SDS or CTAB in the lysis buffer. This protocol also uses glass beads as the DNA precipitation matrix.
3. Circulomics Nanobind CBB Big DNA Kit : initially the recommended extraction protocol for UL sequencing using SQK-ULK001.
4. New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood : a quick, tweakable extraction protocol fitting for a one-day library prep

We optimized the above protocols in human cell lines.

To start DNA extraction, choose one protocol from below:

FindingNemo Extraction 1: Phenol-based Method

FindingNemo Extraction 2: Phenol-free Method

FindingNemo Extraction 3: NEB Monarch Kit

FindingNemo Extraction 4: Circulomics Kit

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:

1. top
2. upper-middle
3. lower-middle
4. bottom

Each sample should be 2.5 μl and combined into a single 2 ml tube.

Add a glass bead and pulse vortex at full speed for a minute.

2400rpm

Quantify and calculate %CV as described in this paper by Koetsier and Cantor.

Next quantify any RNA carry-over using the Qubit RNA Broad Range kit (optional).

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 electrophoresis
• Pulsed-Field Gel Electrophoresis, e.g. , using Pippin Pulse (Sage Science)
• Agilent Bioanalyzer DNA
• Agilent TapeStation DNA

The UL library protocols we have tested are based on ONT's rapid kits, i.e., RAD004 and ULK001, a transposase based method.

We offer three options for library prep protocols with the following features:

1. Modified ULK001 : consistently produces N50 > 100 kb from good input quality UHMW DNA and is our recommended route for best output. The transposase reaction is performed in a large volume of up to 1 ml.
2. Modified RAD004 : also consistently produces N50 > 100 kb from good input quality of UHMW DNA. This can be used when the ligation kit ULK001 is not accessible/available. The transposase reaction is again done in a large volume of up to 1 ml. Considering all the steps in the protocol, the Modified ULK001 kit is more cost effective.
3. KrazyStarFish (KSF; RAD004-based) : can consistently produce N50 > 100 kb with the right transposase to DNA molecule ratio. In addition, it uses filter paper as a matrix for DNA precipitation/clean-up.

Choose one of the protocols we tested below:

FindingNemo Library 1: Modified ULK001

FindingNemo Library 2: Modified RAD004

FindingNemo Library 3: KrazyStarFish (KSF)

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.

Add 1:1 volume of 10 mM Hexamminecobalt(III) Chloride (CoHex) into the DNA solution.

Rotate the tube with a vertical rotator at 9 rpm for 5-10 minutes.

9rpm 0h 5m 0s

Invert the tube 3 times more by hand to ensure the DNA has precipitated and is tightly bound to the beads.

Discard the supernatant. Take care not to disturb the DNA precipitated onto the beads.

Wash the glass beads by gently adding 1 ml of PEGW buffer and gently invert 2-3 times.

Incubate for 3 minutes at room temperature.

Room temperature 0h 3m 0s

Discard most of the supernatant, again taking care not to disturb the DNA precipitate.

Repeat step 13 with 500 μl of the PEGW buffer.

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.

Insert a bead retainer to a collection tube.

Pour the beads from step 16 into the bead retainer and pulse-pin for 1 second in a mini centrifuge (or the shortest time possible) to remove residual wash buffer. Keep the bead retainer.

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.

A	B	C
DNA Input Amount (μg)	No. of glass beads (3-mm diameter)	Elution Buffer Volume (μl)
>30-40	3	225
>20-30	3	180
>5-20	3	120
>2-5	2	90
1-2	1	50

Incubate the library at 37^oC 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 0h 30m 0s

Insert the bead retainer from step 18 into a clean 1.5 ml tube. Pour the beads from step 20 into the bead retainer and centrifuge at 12,000 x g for one minute.

12000rpm

Incubate for at least 30 minutes at room temperature with regular pipette mixing.

Room temperature 0h 30m 0s

Now - you have found Nemo!

Store the library at 4^oC or continue loading it to a flowcell.

4°C

Prime the flow cell as per the MinION or PromethION protocol.

YouTube Video: Priming A MinION Flowcell

Quantify 2-3 μl of the library sample using fluorometric method (Qubit DNA BR kit) or alternatively the spectophotometric method (Nanodrop).

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.

Room temperature 0h 30m 0s

Load the library per SQK-ULK001 protocol and let it tether for another 30 minutes before starting the run.

0h 30m 0s

Select the correct UL sequencing script based on the sequencing kit used (default mux scan should already be set to every 6 hours).

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.

After opening the priming port of the flow cell, check for small bubble under the cover. Draw back a small volume to remove any bubble:

Set a P1000 pipette to 200 μl

Insert the tip into the priming portone

Turn the wheel until the dial shows 220-230 μl, or until a small volume of buffer is seen entering the pipette tip

Using a P1000 pipette, load 400 μl of the NFB plus DNase I into the flow cell priming port.

Close the flow cell priming port and incubate the flow cell in situ for at least 1 hour.

1h 0m 0s

Reprime the flow cell as in step 24.

Reload the library as in step 26-28.