Sep 08, 2021

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

DOI
dx.doi.org/10.17504/protocols.io.bxwrppd6
  • 1University of Nottingham;
  • 2University of British Columbia;
  • 3University of Birmingham
Inswasti Cahyani
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DOI: dx.doi.org/10.17504/protocols.io.bxwrppd6
Protocol Citation: Inswasti Cahyani, John Tyson, Nadine Holmes, Josh Quick, Nicholas Loman, Matthew Loose 2021. FindingNemo: A Toolkit of CoHex- and Glass Bead-based Protocols for Ultra-Long Sequencing on ONT Platforms. protocols.io https://dx.doi.org/10.17504/protocols.io.bxwrppd6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 02, 2021
Last Modified: September 08, 2021
Protocol Integer ID: 52913
Keywords: ultra-long sequencing, cohex, glass bead, nanopore, MinION, UHMW DNA, Monarch, Circulomics, phenol, SDS, CTAB, GM12878, Whatman, PromethION, Nanobind, ULK001, RAD004
Abstract
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:
  1. Extraction
  2. QC
  3. Library preparation
  4. Nemo clean-up using glass beads and Hexamminecobalt(III) Chloride, aka. CoHex.
  5. Flowcell priming and library loading

We have tested and optimised the full protocol in human cell lines.

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.



Guidelines
Acknowledgements

This protocol was developed by Inswasti Cahyani for the Long Read Club with significant contributions from John Tyson and Nadine Holmes, also Josh Quick, and continuous discussion and support of Matt Loose and Nick Loman. We would also like to thank Giron Koetsier (NEB) and Kelvin Liu (Circulomics) for lending their expertise and advance product trials.

Please follow on Twitter for latest updates and results:
@NininUoN
@mattloose

Results

Below are some results from human genomic DNA runs on MinION flowcells, to give an idea of the range of output produced by certain number of input cells:
ABCDEFG
Input CellInput DNA (μg)FRA (μl)Load DNA (μg)N50 (kb)Yield (Gb)Passed Max Read (bp)
6 millions40620142.919.51,692,224
3 million20320139.512.21,611,534
1 million512116.74.01,145,380


Materials
Chemicals/Compounds

  • Reagent5M Ammonium AcetateSigma – AldrichCatalog #A-7330
  • ReagentHexamminecobalt(III) ChlorideAlfa AesarCatalog #A15470
  • ReagentTris-HCl pH 8.0Thermo ScientificCatalog #J22638-AE
  • ReagentEthanol AbsoluteHoneywellCatalog #32221-2.5L
  • ReagentChloroform:Isoamyl Alcohol [24:1]Sigma – AldrichCatalog #25666
  • ReagentJurkat Genomic DNAThermo FisherCatalog #SD1111
  • Reagent40% Polyethylene Glycol MW 8000Sigma – AldrichCatalog #P1458
  • ReagentBuffer-saturated PhenolSigma – AldrichCatalog #77607
  • ReagentIsopropanol AbsoluteFisher ScientificCatalog #P/7500/15
  • Reagent1X Phosphate Buffer SalineFisher ScientificCatalog #15453819
  • ReagentProteinase K, 2mLQiagenCatalog #19131
  • ReagentRNase AQiagenCatalog #19101
  • ReagentDNase I (RNase-free) - 1,000 unitsNew England BiolabsCatalog #M0303S
  • ReagentNuclease-free WaterThermofisherCatalog #AM9920
  • ReagentNaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
  • ReagentEDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G
  • Reagent20% Sodium dodecyl sulfate (SDS)
  • ReagentCetyltrimethylammonium Bromide (CTAB)MP BiologicalsCatalog #02194004-CF
  • ReagentCalcium Chloride
  • ReagentMagnesium ChlorideFisher ScientificCatalog #AC223210010
  • ReagentPotassium Chloride
  • ReagentTriton X-100Sigma AldrichCatalog #T8787-50ML
  • ReagentGlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
  • ReagentHEPESFisher ScientificCatalog #BP310

Made-up Buffers

Tris Lysis Buffer (TLB-SDS or TLB-CTAB)
  • 100 mM NaCl
  • 10 mM Tris-HCl, pH 8.0
  • 25 mM EDTA, pH 8.0
  • TLB-SDS = 0.5% (w/v) SDS or
  • TLB-CTAB = 2% (w/v) CTAB

PEGW Buffer
  • 10% PEG-8000
  • 0.5M NaCl

4x MuA Buffer
  • 100 mM Tris-HCl pH 8.0
  • 40 mM MgCl2
  • 440 mM NaCl
  • 0.2% TritonX-100
  • 40% Glycerol

Nuclease Flush Buffer (NFB)
  • 300mM KCl
  • 2mM CaCl2
  • 10mM MgCl2
  • 15mM HEPES pH 8.0

Kits

  • ReagentNanobind CBB Big DNA KitCirculomicsCatalog #NB-900-001-01 (optional)
  • ReagentMonarch HMW DNA Extraction Kit for Cells & BloodNew England BiolabsCatalog #T3050S (optional)
Note
This kit is necessary for the 1-day protocol

  • ReagentUltra-Long DNA Sequencing Kit (SQK-ULK001)Oxford Nanopore TechnologiesCatalog #SQK-ULK001
Note
Or use the older SQK-RAD004 rapid kit, complemented with 4x MuA buffer
ReagentRapid Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-RAD004


  • ReagentCirculomics Nanobind UL Library Prep KitCirculomicsCatalog #NB-900-601-01 (optional)
  • ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
  • ReagentQubit RNA BR Assay KitThermo Fisher ScientificCatalog #Q10211 (optional)
  • ReagentONT Flow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004 (optional) for multiple loadings and storage on one flow cell)

Disposables

  • ReagentDNA LoBind Tubes, 1.5 mLEppendorfCatalog #0030108051
  • ReagentDNA LoBind 2.0ml PCR Clean Eppendorf TubesEppendorfCatalog #0030 108.078
  • Reagent5Prime Phase-lock Gel (Light) 2 mLVWR InternationalCatalog #733-2477
  • ReagentCentrifuge Tubes with CentriStar™ Cap 15 mlCorningCatalog #10738771
  • Reagent8-strip PCR Tubes with CapsAxygenCatalog #14-222-251
  • ReagentGlass Beads 3 mmScientific Laboratory Supplies LtdCatalog #DD68501 OR ReagentMonarch DNA Capture BeadsNew England BiolabsCatalog #T3005L
  • ReagentThin-wall PCR Tubes 0.5 mlFisher ScientificCatalog #12194142
Note
cut tube 2-3 mm from the bottom to make a bead retainer
OR ReagentMonarch Bead RetainersNew England BiolabsCatalog #T3004L
  • ReagentMonarch Collection Tubes II - 100 tubesNew England BiolabsCatalog #T2018L (optional)
Note
or use any 1.5 ml centrifuge tube as collection tube

  • ReagentWhatman Filter Paper No.3Catalog #SKU1153211
Note
KrazyStarFish (KSF) disk:
6 mm star-shaped (or round-shaped) disk punched out of Whatman #3 cellulose filter paper

  • Wide-bore (or cut off) P1000 and P200 tips



































Safety warnings
When handling phenol always wear PPE, keep a solution of 50% (w/v) PEG-400 nearby to treat the burn in the case of accidental splashes.
Before start
Things to observe at all times
  • Excessive and vigorous pipetting and vortexing should be avoided as these may shear the DNA.
  • Make up buffers with nuclease-free water to avoid introducing nucleases to solutions.
  • Avoid unnecessary heating and freezing; isolated DNA should be stable for storage in the fridge for months.
UHMW DNA Extraction
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:
  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:

Protocol
FindingNemo Extraction 1: Phenol-based Method
NAME

FindingNemo Extraction 1: Phenol-based Method

CREATED BY
Inswasti Cahyani

Optional
This protocol is a scaled-down and modified version of the “Ultra-long read sequencing protocol for RAD004 V.3” by Josh Quick (https://dx.doi.org/10.17504/protocols.io.mrxc57n).
Note
Elution volume is adjusted for downstream application of preparing ultra-long DNA library following the new ONT protocol (SQK-ULK001).

Pellet 5 million cells in a 2 ml tube by centrifuging at 500 x g for 5 min at 4oC.
Centrifigation500 x g, 4°C, 00:05:00

5m
Wash with 500 μl cold PBS and centrifuge at 500 x g for 3 min at 4oC. Discard supernatant.
Centrifigation500 x g, 4°C, 00:03:00

3m
Resuspend well by pipette mixing in 20 μl cold PBS.
Note
Thorough resuspension is important for next lysis step.

Add 1 ml TLB-SDS and 100 μg RNase A (1 μl) and vortex at full speed for 5 seconds.
Shaker2000 rpm, 00:00:05
Incubate at 37°C for 1 hour, mix by inversion every 15 minutes.
Temperature37 °C Duration01:00:00 mix per 15 min
1h
Add 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times.
Incubate at 50°C for 2 hours, mix every 30 minutes by slow inversion 3 times.
Temperature50 °C Duration02:00:00 mix per 30 min (3x inversion)

2h
Split the lysate into 2 phase-lock gel tubes (ca. 550 ul per tube).
Add 550 μl buffer-saturated phenol to each tube containing lysate.
Place on a HulaMixer or any vertical rotator at 20 rpm for 10 minutes. If a fine emulsion has not formed after a minute gradually increase the rotation speed.

Duration00:10:00 vertical rotator

10m
Centrifuge at 4500 rpm for 10 minutes.
Centrifigation4500 rpm, Room temperature, 00:10:00

10m
Transfer the aqueous phase to another phase-lock gel tube by pouring or using a wide-bore P1000 tip.
Add 250 μl buffer-saturated phenol and 250 μl chloroform-isoamyl alcohol to each tube.
Repeat step 11-12 and continue to step 15.
Go togo to step #1.1.11 Go togo to step #1.1.12

Transfer and combine the aqueous phase to a 2 ml tube (sample will be ca. 1 ml).
Add 1 ml chloroform-isoamyl alcohol.
Repeat step 11-12 and continue to 17.
Go togo to step #1.1.11 Go togo to step #1.1.12

Using a wide-bore P1000 tip, transfer the aqueous phase to a 5 ml conical tube.
Note
Do not bring any liquid from the interface of the chloroform phase, as this will affect DNA purity in the downstream steps.

Add 0.4x volume of 5M Ammonium Acetate (ca. 400 μl). Mix by slow inversion of tube.
Add 3 clean glass beads.
Add 3 ml absolute ethanol.
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes.
Duration00:05:00 vertical rotator
Note
If a rotator is not available, hand inversion for 30-40 times can be used. Invert the tube slowly by hand so that a full inversion cycle takes 5-6 seconds.


5m
Remove solution, taking care not to disturb bound DNA on the glass beads.
Wash bound DNA with 1 ml of 70% ethanol. Invert tube for 2-3 times and discard the ethanol.
Repeat step 23.
Insert a bead retainer to a collection tube.
Pour the beads into the bead retainer and spin for 1 s 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 250 µl of elution buffer.
Note
Do not let the beads with DNA dry out. (As an alternative, 250 μl of elution buffer can be aliquoted into a 2 ml tube prior to this step.)

Critical
Incubate at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P1000 tip to aid elution.
Temperature37 °C Duration00:30:00 mix per 10 min

30m
Insert the bead retainer from step 25 into a clean 2 ml DNA low-bind tube.
Pour the beads from step 27 and centrifuge at 12,000 x g for 1 minute.
Centrifigation12000 rpm, Room temperature, 00:01:00

1m
Add another 510 μl of elution buffer to the eluate from step 28 and mix with a wide-bore P1000 tip.
Leave overnight at room temperature.
TemperatureRoom temperature DurationOvernight
Note
DNA sample will be viscous, but it is important to pipette thoroughly with a wide-bore pipette tip to ensure homogenization.
Homogeneous DNA sample will ensure consistent output length and yield.

Quantify DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values. If the DNA is not sufficiently homegeneous, incubate the DNA for longer.
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001".
If only SQK-RAD004 is available, follow library preparation in Section "Modified RAD004" or "KrazyStarFish (KSF)".
Temperature4 °C for storage


Protocol
FindingNemo Extraction 2: Phenol-free Method
NAME

FindingNemo Extraction 2: Phenol-free Method

CREATED BY
Inswasti Cahyani

Optional
This protocol is adapted from Monarch® HMW DNA Extraction Kit for Cells & Blood.
Note
Either SDS (anionic surfactant) or CTAB (cationic surfactant) can be used in the lysis buffer.
Providing alternative surfactants in the lysis buffer may help with different cell systems that require different biochemistry.

Pellet 3 million cells in 1.5 ml tube by centrifuging at 1000 x g for 1 min at 4oC.
Centrifigation1000 x g, 4°C, 00:01:00

1m
Wash with PBS (make sure all media and serum are rinsed off), spin at 1000 x g for 1 min at 4oC.
Centrifigation1000 x g, 4°C, 00:01:00
1m
Add 149 μl of TLB-SDS or TLB-CTAB which has been added with 100 μg RNaseA (1 μl) and vortex at full speed for 3 seconds.
Note
Make master mix of the TLB and RNaseA if handling more than one sample.

Incubate at 37°C for 10 minutes.
Temperature37 °C Duration00:10:00

10m
Add 140 μl TLB and 200 μg Proteinase K (10 μl). Mix by slow inversion 5 times or with a P1000 wide-bore pipette tip.
Incubate at 55°C for 20 minutes.
Temperature55 °C Duration00:20:00
Note
Shaking in a thermomixer at 300-700 rpm can help lysis and homogenization, especially when more than 3 million cells are used.



20m
Add 75 μl of 5M Ammonium acetate, mix well with wide-bore P1000 pipette tip.
Add 3 clean glass beads to the cell lysate.
Add 275 μl of Isopropanol
Rotate the tube with a vertical rotator at 9 rpm for 5 minutes.
Duration00:05:00 vertical rotator
Note
If a rotator is not available, hand inversion for 30-40 times can be used. Invert the tube slowly by hand so that a full inversion cycle takes 4-5 seconds.


5m
Remove and discard liquid by pipetting.
Wash bound DNA with 1 ml of 70% ethanol, invert tube 3 times, remove and discard ethanol.
Repeat step 13 once with 500 μl. Discard the ethanol, taking care not to disturb the DNA precipitate.
Insert a bead retainer to a collection tube.
Pour the beads into the bead retainer and spin for 1 s 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 250 µl of elution buffer.
Note
Do not let the beads with DNA dry out. (As an alternative, 250 μl of elution buffer can be aliquoted into a 2 ml tube prior to this step.)

Critical
Incubate at 37oC for 30 min. Gently aspirate and dispense the eluate over the glass beads at regular intervals with a wide-bore P1000 tip to aid elution.
Temperature37 °C Duration00:30:00 mix per 10 min

30m
Insert the bead retainer from step 15 into a clean 2 ml DNA low-bind tube.
Pour the beads from step 17 and centrifuge at 12,000 x g for 1 minute.
Centrifigation12000 rpm, Room temperature, 00:01:00

1m
Quantify DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values. If the DNA is not sufficiently homegeneous, incubate the DNA for longer.
Store at 4oC or continue to UL Library Preparation as per "Modified ULK001".
If only SQK-RAD004 is available, follow library preparation in "Modified RAD004" or "KrazyStarFish (KSF)".
Temperature4 °C for storage


Protocol
FindingNemo Extraction 3: NEB Monarch Kit
NAME
FindingNemo Extraction 3: NEB Monarch Kit
CREATED BY
Inswasti Cahyani

Optional
This protocol is using Monarch® HMW DNA Extraction Kit for Cells & Blood.
ReagentMonarch HMW DNA Extraction Kit for Cells & BloodHoneywell FlukaCatalog #T3050S

We have obtained optimal 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.
Note
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.

To complete DNA extraction from cell to library prep and sequencing in one day, start early!
Note
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.


Dilute the eluted DNA with 150 μl of NEB Elution Buffer II.
Note
  • This is following the NEB UHMW Monarch kit protocol where DNA is first eluted with 100 μl buffer. After this step, sample volume will be 250 μl total.
  • Quantification of a very viscous UHMW DNA is problematic and will not produce accurate results, hence the dilution.
  • Gradual dilution is recommended to achieve homogeneous concentration of 50-100 ng/μl.

Incubate the eluted DNA at 37oC for about 2-3 hours with regular pipette mixing.
Temperature37 °C Duration03:00:00 max (2-3 hours)
Note
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.

3h
Quantify DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values. If the DNA is not sufficiently homegeneous, incubate the DNA for longer.
Store at 4oC or continue to UL Library Preparation as per "Modified ULK001".
If only SQK-RAD004 is available, follow library preparation as in "Modified RAD004" or "KrazyStarFish (KSF)".
Temperature4 °C for storage


Protocol
FindingNemo Extraction 4: Circulomics Kit
NAME
FindingNemo Extraction 4: Circulomics Kit
CREATED BY
Inswasti Cahyani

Optional
This protocol is using the Nanobind CBB Big DNA Kit (see Materials).
Follow the manufacturer’s instructions as described here.
After overnight incubation at room temperature, quantify the DNA as per "UHMW DNA QC" and check homogeneity by calculating %CV values.
Store at 4oC or continue to UL Library Preparation as per Section "Modified ULK001".
If only SQK-RAD004 is available, follow library preparation as in Section "Modified RAD004" or "KrazyStarFish (KSF)".
Temperature4 °C for storage

UHMW DNA QC
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:
  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.
Note
  • 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.

Add a glass bead and pulse vortex at full speed for a minute.
Shaker2400 rpm, 00:01:00 vortex max speed

Quantify and calculate %CV as described in this paper by Koetsier and Cantor.
Note
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).
ReagentQubit dsDNA BR (Broad Range) assayThermo Fisher ScientificCatalog #Q32850
ReagentJurkat Genomic DNAThermo FisherCatalog #SD1111


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

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
Optional
UL Library Prep
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:
  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:

Protocol
FindingNemo Library 1: Modified ULK001
NAME

FindingNemo Library 1: Modified ULK001

CREATED BY
Inswasti Cahyani

Extracted UHMW DNA is often difficult to quantify due to its viscosity. However, accurate measurement of DNA concentration is crucial for calculating optimum ratio of the transposase enzyme to the DNA molecules.
We provide a protocol section for quantifying UHMW DNA in our 'FindingNemo' protocol master file.
Properly quantified DNA can then be processed for this library prep.
Both cell number and DNA concentration/amount are used to calculate the amount of transposase (FRA) and adapter (RAP-F).

We follow the original SQK-ULK001 protocol for the optimum ratio of transposase amount to human genomic DNA:
6 μl FRA to 6 million human cells (or around 40 μg DNA)

For other species, the genome size has to be taken into account and the FRA to DNA ratio optimised, e.g., we had optimised a non-human cell line of 6.2 Gb genome at:
2.5 μl FRA to 1 million non-human cell (around 12-15 μg DNA)
In a 2 ml tube, dilute UHMW DNA to a concentration of around 50 ng/μl in a total volume of 750 μl (with water or elution buffer if required).
Mix well with a P1000 wide-bore tip.
Note
  • DNA concentration can still range from 20-50 ng/μl to have optimum tagmentation reaction.
  • If input DNA amount is less than 20 μg (1-3 million cells used), halve all the reaction volumes, i.e., 375 μl total DNA volume instead of 750 μl as in the table below.
  • It is important to have as homogeneous DNA as possible at this step so the transposase can access and cut the DNA solution with an even distribution. It is OK to pipette thoroughly but gently.

ABCDE
Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)
6>20-4075020-501000
5
4
35-20375500
2
1

Critical
In a 1.5 ml tube, dilute the corresponding amount of transposase (FRA) with the dilution buffer (FDB) to a total volume of 250 μl (or 125 μl if doing half-reaction). More details in the table below.

ABCDE
Cell No. (million)Approx. DNA amount (μg)FRA (μl)FDB (μl)Total reaction volume (μl)
6>20-4062441000
55245
44246
35-203122500
22123
11124

Mix the diluted FRA by vortexing for 2-3 seconds.
Using a P1000 wide-bore tip, add the diluted FRA to the DNA sample.
Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.
Mix thoroughly by gentle pipetting.
TemperatureOn ice

Incubate the reaction as follows:
Temperature23 °C Duration00:10:00
Temperature70 °C Duration00:05:00
TemperatureRoom temperature Duration00:10:00 at least
Note
It is important that the room temperature at the fragmentation step (first incubation step) does not fall below 20oC to ensure optimum reaction condition. The use of a water bath or heating block is recommended.

25m
Critical
Add the corresponding volume of sequencing adapter (RAP-F) as in the table below.

ABCDE
Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP-F (μl)Total reaction volume (μl)
6>20-40651000
554.2
443.3
35-2032.5500
221.7
110.8


Note
  • Use a P1000 wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed.
  • Tube inversion can be used to aid mixing.


Incubate for 30 minutes at 23oC.
Temperature23 °C Duration00:30:00

30m

Protocol
FindingNemo Library 2: Modified RAD004
NAME
FindingNemo Library 2: Modified RAD004
CREATED BY
Inswasti Cahyani

Optional
Extracted UHMW DNA is often difficult to quantify due to its viscosity. However, accurate measurement of DNA concentration is crucial for calculating optimum ratio of the transposase enzyme to the DNA molecules.
We provide a protocol section for quantifying UHMW DNA in our 'FindingNemo' protocol master file.
Properly quantified DNA can then be processed for this library prep.
Both cell number and DNA concentration/amount are used to calculate the amount of transposase (FRA) and adapter (RAP).
We base this protocol on SQK-RAD004 but follow the SQK-ULK001 protocol for the optimum ratio of transposase amount to human genomic DNA:
6 μl FRA to 6 million human cells (or around 40 μg DNA)

For other species, the genome size has to be taken into account and the FRA to DNA ratio optimised, e.g., we had optimised a non-human cell line of 6.2 Gb genome at:
2.5 μl FRA to 1 million non-human cell (around 12-15 μg DNA)
The following table lists the replaced reagents between ULK001 and RAD004.
ABC
ReagentULK001RAD004 (modified)
Dilution bufferFDBMuA buffer (see Materials)
TransposaseFRA*FRA^
Sequencing adapterRAP-FRAP
(*) FRA enzyme in ULK001 kit
(^) FRA enzyme in RAD004 kit

In a 2 ml tube, dilute UHMW DNA to a concentration of around 50 ng/μl in a total volume of 750 μl (with water or elution buffer if required).
Mix well with a P1000 wide-bore tip.
Note
  • DNA concentration can still range from 20-50 ng/μl to have optimum transposase reaction.
  • If input DNA amount is less than 20 μg (1-3 million cells used), halve all the reaction volumes, i.e., 375 μl total DNA volume instead of 750 μl as in the table below.
  • It is important to have as homogeneous DNA as possible at this step so the transposase can access and cut the DNA molecules with an even distribution. It is OK to pipette thoroughly but gently.

ABCDE
Cell No. (million)Approx. DNA amount (μg)Total DNA volume (μl)DNA concentration (ng/μl)Total reaction volume (μl)
6>20-4075020-501000
5
4
35-20375500
2
1

Critical
In a 1.5 ml tube, dilute the corresponding amount of transposase (FRA) with MuA buffer to a total volume of 250 μl (or 125 μl if doing half-reaction). More details in the table below.

ABCDE
Cell No. (million)Approx. DNA amount (μg)FRA (μl)4X MuA Buffer (μl)Total reaction volume (μl)
6>20-4062441000
55245
44246
35-203122500
22123
11124

Mix the diluted FRA by vortexing for 2-3 seconds.
Using a P1000 wide-bore tip, add the diluted FRA to the DNA sample.
Stir the reaction with the pipette tip whilst expelling the diluted FRA to ensure an even distribution.
Mix thoroughly by gentle pipetting.
TemperatureOn ice

Incubate the reaction as follows:
Temperature23 °C Duration00:10:00
Temperature70 °C Duration00:05:00
TemperatureRoom temperature Duration00:10:00 at least
Note
It is important that the room temperature at the fragmentation step (first incubation step) does not fall below 20oC to ensure optimum reaction condition. The use of a water bath or heating block is recommended.

Critical
Add the corresponding volume of sequencing adapter (RAP) as in the table below.

ABCDE
Cell No. (million)Approx. DNA amount (μg)FRA (μl)RAP (μl)Total reaction volume (μl)
6>20-40651000
554.2
443.3
35-2032.5500
221.7
110.8

Note
  • Use a P1000 wide-bore tip to pipette mix. Visually check to ensure the reaction is thoroughly mixed.
  • Tube inversion can be used to aid mixing.

Incubate for 30 minutes at 23oC.
Temperature23 °C Duration00:30:00


Protocol
FindingNemo Library 3: KrazyStarFish (KSF)
NAME

FindingNemo Library 3: KrazyStarFish (KSF)

CREATED BY
Inswasti Cahyani

Optional
This library prep is based on the rapid kit (SQK-RAD004) and using a home-made MuA buffer (see Materials).
Input DNA is based on its concentration/amount without requiring prior knowledge on input cell number.
In a 1.5 ml tube (labelled as DNA), gently mix 75 μl DNA (~100 ng/μl) with 25 μl 4x MuA buffer.
Note
Standard input DNA is 7.5 μg and can be for three loadings on MinION.

In another 1.5 ml tube (labelled as MuA), mix 25 μl 4x MuA buffer with 74 μl water and 0.1-1 μl FRA (depending on N50 targeted).
Note
We consistently obtained N50 >= 100 kb using a ratio of:
0.6 μl FRA per 10 μg human genome DNA

Add/mix the 100 μl content of the MuA tube into the DNA containing tube, pipetting slowly with a wide-bore P200 tip and moving the tip constantly.
Make 100 μl aliquots into PCR tubes and treat at 30oC for 1 min, 80oC for 1 min and then cool to room temperature.
Temperature30 °C Duration00:01:00
Temperature80 °C Duration00:01:00
TemperatureRoom temperature cool-off

Pool reactions into a single tube and remove ~190 μl into a fresh 1.5 ml tube.
Add 12 μl of 5 M NaCl and mix gently (by flicking or pipetting with a wide-bore P200 tip).
Add a “kRAZYsTARFISH” filter (see Materials) to the tube so it is submerged.
Add 142 μl Isopropanol to the tube.
Mix contents gently by inversion 20-30 times, allowing UHMW DNA to collect and condense onto the filter.
Pulse-spin the tube and remove the liquid by passing pipette tip past the filter.
Note
If the filter sticks to the side of the tip during this process gently place it back onto the side of the tube.

Wash the filter by addition of 500 μl 70% ethanol and gently invert the tube a few times.
Pulse-spin the tube and remove the liquid leaving the filter behind in the tube.
Repeat the 70% ethanol wash (step 12) once.
Pulse-spin the tube and remove any residual liquid leaving the filter behind and allow to air dry for a couple of minutes.
Transfer the filter to a 2 ml tube by tipping.
Add 125 μl of EB buffer, covering the filter, and allow tagged DNA to resuspend for 20-30 mins at 37oC with occasional gentle mixing (flicking or pipetting with a wide-bore P200 tip).
Temperature37 °C Duration00:30:00 max
Note
We have found efficiency of resuspension to be very good from the cellulose filter, but some gains may be had if library is left longer when using larger amounts of UHMW DNA as input.

Quantify as per section "UHMW DNA QC".
Store sample at 4oC until ready to proceed to adapter (RAP) addition followed by sequencing.
This will be sufficient for about 3 sequencing runs/reloads.
Temperature4 °C for storage

Remove 37.0 μl of tagged DNA library into a fresh 1.5 ml tube.
Add 37.5 μl SQB buffer, mix gently (by flicking or pipetting with a wide-bore P200 tip).
Add 0.5 μl RAP, mix gently and incubate at room temperature for 30 minutes.
TemperatureRoom temperature Duration00:30:00
Continue to Section "Flowcell Priming & Library Loading", or an optional "Nemo" clean-up step.
Note
Adding the Nemo clean-up step will remove most free adapters. In our experience testing this, the yield/occupancy was only slightly improved as this parameter depends more on the optimum transposase reaction. Using the Nemo clean-up may improve non-optimal transposase-cut library.

NEMO Library Clean-up
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.
Note
  • 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.

Add 1:1 volume of 10 mM Hexamminecobalt(III) Chloride (CoHex) into the DNA solution.
Note
When the clean-up follows ONT ultra-long library preparation (SQK-ULK001), volume can typically range from 500-1000 μl.

Rotate the tube with a vertical rotator at 9 rpm for 5-10 minutes.
Shaker9 rpm vertical rotator Duration00:05:00 10m max
Note
  • 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.


5m
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.
TemperatureRoom temperature Duration00:03:00

3m
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.

Note
Omitting Dry-spin Step
When 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.

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.

ABC
DNA Input Amount (μg)No. of glass beads (3-mm diameter)Elution Buffer Volume (μl)
>30-403225
>20-303180
>5-203120
>2-5290
1-2150

Note
  • 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.

Critical
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.
Temperature37 °C Duration00:30:00 mix per 10 min

30m
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.
Centrifigation12000 rpm, Room temperature, 00:01:00

1m
Incubate for at least 30 minutes at room temperature with regular pipette mixing.
TemperatureRoom temperature Duration00:30:00 at least

30m
Now - you have found Nemo!


Store the library at 4oC or continue loading it to a flowcell.
Temperature4 °C for storage
Note
Load at least 1 μg for MinION sequencing (or all of the library for input cells of 1 million).


Flowcell Priming & Library Loading
Flowcell Priming & Library Loading
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.
TemperatureRoom temperature Duration00:30:00

30m
Load the library per SQK-ULK001 protocol and let it tether for another 30 minutes before starting the run.
Duration00:30:00 tethering
30m
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)
Home-brew Flowcell Wash/Flush (Optional)
1h
1h
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.
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:
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.
Duration01:00:00 at least

1h
Reprime the flow cell as in step 24.
Go togo to step #24

Reload the library as in step 26-28.
Go togo to step #26 Go togo to step #27 Go togo to step #28