TITLE: FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms
AUTHORS: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection of protocols is designed to enable ultra-long (UL) reads on Nanopore sequencers. It is split into five sections dealing with ultra-high molecular weight (UHMW) DNA:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Extraction</li><li style = "counter-reset:ol0;">QC </li><li style = "counter-reset:ol0;">Library preparation </li><li style = "counter-reset:ol0;">Nemo clean-up using glass beads and Hexamminecobalt(III) Chloride, aka. CoHex. </li><li style = "counter-reset:ol0;">Flowcell priming and library loading</li></ol></div><br/><div class = "text-block"><span>We have tested and optimised the full protocol in </span><span style = "font-weight:bold;">human cell lines</span><span>. </span></div><br/><div class = "text-block">Various options are available for each of the steps and we hope that the components here will be useful to the community and provide a long read toolkit.</div><br/><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/ech79wh6.jpg" /></div><br/></div>

[STEPS]
?. [UHMW DNA Extraction]
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: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.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.Circulomics Nanobind CBB Big DNA Kit: initially the recommended extraction protocol for UL sequencing using SQK-ULK001.New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood: a quick, tweakable extraction protocol fitting for a one-day library prepWe optimized the above protocols in human cell lines.To start DNA extraction, choose one protocol from below:
?. [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 DNA sample and if the sample is 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 a 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 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]
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: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.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.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:
?. [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 19). 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 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), volume 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 the 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 2-3 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 13 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 to a collection tube.
?. [NEMO Library Clean-up]
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.
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 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.
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 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 portone
?. [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 24.
?. [Home-brew Flowcell Wash/Flush (Optional)]
Reload the library as in step 26-28.
?. [UHMW DNA Extraction]
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?. [UHMW DNA Extraction]
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?. [UHMW DNA Extraction]
{"blocks":[{"key":"37jk","text":"This is a sub-protocol designed to extract\/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood. ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":74,"offset":151,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"3bndd","text":"A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"9jbg1","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"fuv0f","text":"The New England Biolabs (NEB) Monarch HMW DNA Extraction Kit for Cells & Blood is a quick, tweakable extraction protocol fitting for one-day library prep.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":75,"offset":4,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"767re","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"155p7","text":"We tested this sub-protocol in human cell line, with input cells varied between 1-6 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":15,"offset":31,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"1g7k1","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [UHMW DNA Extraction]
{"blocks":[{"key":"37jk","text":"This is a sub-protocol designed to extract\/isolate ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers using the Circulomics Nanobind CBB Big DNA Kit. ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":36,"offset":155,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"3bndd","text":"A DNA extraction protocol that yields clean and homogeneous UHMW DNA is important for a good UL sequencing output. The choice of protocol should be based on achieving these parameters.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"9jbg1","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"fuv0f","text":"The Circulomics Nanobind CBB Big DNA Kit was initially the recommended extraction protocol for UL sequencing using SQK-ULK001.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":36,"offset":4,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"3ivon","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"155p7","text":"We tested this sub-protocol in human cell line, with standard input cells of 6 millions. As a rule of thumb, a million cells will suffice for one load on a MinION.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":15,"offset":31,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"1g7k1","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [UL Library Prep]
{"blocks":[{"key":"37jk","text":"This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocols we tested are based on ONT's rapid kit, i.e., SQK-ULK001, a transposase based adapter ligation kit.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":6,"offset":224,"style":"italic"},{"length":10,"offset":230,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"e5d50","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"7g7ps","text":"Modified ULK001 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA and is our recommended route for best output as it is also the most-cost effective. ","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":16,"offset":0,"style":"bold"},{"length":21,"offset":105,"style":"italic"}],"entityRanges":[],"data":[]},{"key":"96uj3","text":"Transposase-based reaction is done in a large volume of up to 1 ml.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"bh4ji","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"bdtq5","text":"The working principle of the ULK001 protocol is shown in the diagram below:","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"bv8ng","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"2ghud","text":"  ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":0,"length":1,"offset":0}],"data":[]},{"key":"9qa3j","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[{"type":"image","data":{"guid":"1DDB97400BD611EC87ADA362449CCFD9","height":210,"id":133353,"is_video":false,"legend":{"blocks":[{"data":[],"depth":0,"entityRanges":[{"key":0,"length":59,"offset":8}],"inlineStyleRanges":[],"key":"43fb3","text":"source: https:\/\/store.nanoporetech.com\/uk\/rapid-sequencing-kit.html","type":"unstyled"}],"entityMap":[{"data":{"guid":"6A61FFB70CA711ECA7CD0A58A9FEAC02","url":"https:\/\/store.nanoporetech.com\/uk\/rapid-sequencing-kit.html"},"type":"link"}]},"mime":"image\/png","nestedSelector":null,"original_name":"rapid.png","placeholder":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echr7t5n.jpg","shadow":false,"source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echj7t5n.jpg","webp_placeholder":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echm7t5n.webp","webp_source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echi7t5n.webp","width":240}}]}
?. [UL Library Prep]
{"blocks":[{"key":"37jk","text":"This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":6,"offset":227,"style":"italic"},{"length":10,"offset":233,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"e5d50","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"7g7ps","text":"This Modified RAD004 protocol consistently produced N50 > 100 kb from a good input quality of UHMW DNA, for when ULK001 is not accessible\/available. Transposase-based reaction is done in a large volume of up to 1 ml.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":15,"offset":5,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"eaq5v","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"e0c32","text":"The working principle of the RAD004 kit is shown in the diagram below:","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"bbq1j","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"ep7i9","text":"  ","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[{"key":0,"length":1,"offset":0}],"data":[]},{"key":"528h9","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[{"type":"image","data":{"guid":"8BD28F400BD311EC9F7E87981D1EA383","height":228.24999999999991,"id":133360,"is_video":false,"legend":{"blocks":[{"data":[],"depth":0,"entityRanges":[{"key":0,"length":59,"offset":8}],"inlineStyleRanges":[],"key":"fmh6v","text":"source: https:\/\/store.nanoporetech.com\/uk\/rapid-sequencing-kit.html","type":"unstyled"}],"entityMap":[{"data":{"guid":"8C3A46D20CA711ECA7CD0A58A9FEAC02","url":"https:\/\/store.nanoporetech.com\/uk\/rapid-sequencing-kit.html"},"type":"link"}]},"mime":"image\/png","nestedSelector":null,"original_name":"rapid.png","placeholder":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echv7t5n.jpg","shadow":false,"source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echs7t5n.jpg","webp_placeholder":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echu7t5n.webp","webp_source":"https:\/\/s3.amazonaws.com\/protocols-files\/files\/echp7t5n.webp","width":260.85714285714283}}]}
?. [UL Library Prep]
{"blocks":[{"key":"37jk","text":"This sub-protocol is designed to prepare library from extracted ultra-high molecular weight (UHMW) DNA to obtain ultra-long (UL) reads on Nanopore sequencers. The UL library protocol we tested here is based on ONT's rapid kit, i.e., SQK-RAD004, a transposase based adapter ligation kit.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":6,"offset":227,"style":"italic"},{"length":10,"offset":233,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"e5d50","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"7g7ps","text":"We named this protocol KrazyStarFish (KSF). It offers a different approach to UL library prep, by using filter paper shaped as a starfish at the DNA precipitation\/clean-up step. It can consistently produced N50 > 100 kb with the right transposase to DNA ratio.","type":"unstyled","depth":0,"inlineStyleRanges":[{"length":19,"offset":23,"style":"bold"}],"entityRanges":[],"data":[]},{"key":"39olq","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]},{"key":"8ltkj","text":"This protocol was developed by John Tyson at UBC, Vancouver.","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}