Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)

Oxford Nanopore Technologies
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MinION: Protocol

Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) V ULK_9177_v114_revP_11Dec2024

  • Extraction of ultra-high molecular weight (uHMW) gDNA
  • Reliably generate and sequence ultra-long read length N50s (>50 kb)
  • High yield
  • Compatible with R10.4.1 flow cells

For Research Use Only

This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.

FOR RESEARCH USE ONLY

概要

  • Extraction of ultra-high molecular weight (uHMW) gDNA
  • Reliably generate and sequence ultra-long read length N50s (>50 kb)
  • High yield
  • Compatible with R10.4.1 flow cells

For Research Use Only

This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.

Document version: ULK_9177_v114_revP_11Dec2024

1. Overview of the protocol

重要

This is an Early Access product

For more information about our Early Access programmes, please see this article on product release phases.

Please ensure you always use the most recent version of the protocol.

Introduction to the Ultra-Long DNA Sequencing Kit protocol (SQK-ULK114)

This protocol describes the complete workflow from extracting gDNA from frozen tissue or purified cells from whole blood to the sequencing of ultra-high molecular weight (uHMW) gDNA using the Ultra-Long DNA Sequencing Kit (SQK-ULK114). We have also included the procedure to isolate white blood cells (WBCs) from whole blood and how to quantify gDNA developed by Paul A ‘Giron’ Koetsier & Eric J Cantor, 2021.

We have used the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060) to extract the uHMW gDNA for both input types when developing this protocol. Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.

Per reaction, there is enough library generated for multiple consecutive loads onto a flow cell to increase output. To load a library six times on a MinION/GridION flow cell, a flow cell wash is required to recover channels.

Steps in the sequencing workflow: Prepare for your experiment You will need to:

  • If working with whole blood, isolate white blood cells. If working with frozen tissue, isolate cells from the tissue
  • Extract your uHMW gDNA
  • Quantify your sample
  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • If not already installed, download the software for acquiring and analysing your data
  • Check your flow cell(s) to ensure it has enough pores for a good sequencing run

Library preparation You will need to:

  • Tagment your DNA using a diluted fragmentation mix
  • Attach Rapid Adapters to the DNA ends
  • Clean-up your sample by precipitating your DNA and elute overnight
  • Prime the flow cell and load your DNA library into the flow cell

ULK114 workflow V1-3


Sequencing and analysis You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
  • Optional: Start the EPI2ME software and select a workflow for further analysis

Flow cell loading and flushing

The Ultra-Long DNA Sequencing Kit (SQK-ULK114) protocol generates viscous DNA which can affect flow cell loading. We have modified the flow cell loading steps to take account for this. Please take care and follow the steps carefully to avoid damaging the flow cell.

To increase output, we recommend loading an ultra-long library three times per flow cell. A flow cell wash using the Flow Cell Wash Kit (EXP-WSH004) is required between each subsequent library load to recover channels. To run a second library straight away, please follow the modified method in this protocol: To run another library of ultra-long DNA on a MinION/GridION flow cell straight away.

Best practice for handling uHMW gDNA

When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.

To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.

While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.

For further information, please refer to the troubleshooting section.

重要

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

材料
  • Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
  • Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
  • Flow Cell Wash Kit (EXP-WSH004)
消耗品
  • MinIONとGridIONのFlow Cell
  • Isopropanol, 100% (Fisher, 10723124)
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 5 ml Eppendorf DNA LoBind tubes
  • 15 ml Falcon tubes
装置
  • MinIONかGridION のデバイス
  • MinIONとGridIONのFlow Cell ライトシールド
  • Thermal cycler or heat block
  • Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
  • ボルテックスミキサー
  • Microfuge
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • Qubit fluorometer (or equivalent)
  • Ice bucket with ice
  • Timer
重要

The above list of materials, consumables, and equipment is for the library preparation section of the protocol. Depending on the sample type, additional reagents will be needed for sample processing and DNA extraction; these are listed in the "Sample preparation" section of the protocol.

For this protocol, you will need to extract gDNA from 6 million cells in 40 µl PBS before starting the library preparation.

This protocol has been developed using the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060). Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.

This method has been validated for use on the following inputs:

  • 6 million white blood cells isolated from 1.6 ml blood (bovine), using RBC Lysis Solution (QIAGEN, cat # 158904)
  • 6 million cells isolated from 1 g frozen tissue, using pluriSelect Cell Straining equipment.

フローセルのチェックをしてください

シークエンシング実験を開始する前に、フローセルのポアの数を確認することを強くお勧めします。このフローセルの確認は、MinION/GridION/PromethIONの場合は代理店への到着から１２週間以内に行ってください。またはFlongle Flow Cellの場合は代理店への到着から4週間以内に行う必要があります。Oxford Nanopore Technologiesは、フローセルチェックの実施から2日以内に結果が報告され、推奨される保管方法に従っていた場合に、以下の表に記載されているナノポアの有効数に満たさない場合には、フローセルを交換します。 フローセルのチェックを行うには、Flow Cell Check documentの指示に従ってください。

Flow cell 保証する最小有効ポア数（以下の数未満のフローセルが交換対象となります）
Flongle Flow Cell 50
MinION/GridION Flow Cell 800
PromethION Flow Cell 5000

Ultra-Long DNA Sequencing Kit (SQK-ULK114) contents

We had previously uncovered an issue with the Precipitation Stars (PS) found in the SQK-ULK114 kits and removed their use from the method to prevent any potential issues.

We have since improved our manufacturing and internal validation processes of the Precipitation Stars (PS) and are now in the position to reintroduce their use with the SQK-ULK114 Kits.

Kit format with improved precipitation stars that can be used:

Batch ULK114.30.0001 or newer

ULK114 Kit format with improved Precipitation stars

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
Rapid Adapter RA Green 1 40
Fragmentation Mix FRA Amber 1 50
FRA Dilution Buffer FDB Clear 1 1,600
Elution Buffer EB Black 2 1,500
Extraction EB EEB Orange 3 1,700
Sequencing Buffer UL SBU Red 2 1,000
Loading Solution UL LSU White cap, pink label 1 200
Flush Tether UL FTU Purple 1 600
Flow Cell Flush FCF Blue 2 15,500
Precipitation Buffer PTB Blue 2 1,700
Precipitation Star PS Yellow 6 1 star

Kit format where stars should not be used:

Batch ULK114.20.xxxx or older:

ULK114 tubes

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
Rapid Adapter RA Green 1 40
Fragmentation Mix FRA Amber 1 50
FRA Dilution Buffer FDB Clear 1 1,600
Elution Buffer EB Black 2 1,500
Extraction EB EEB Orange 3 1,700
Sequencing Buffer UL SBU Red 2 1,000
Loading Solution UL LSU White cap, pink label 1 200
Flush Tether UL FTU Purple 1 600
Flow Cell Flush FCF Blue 2 15,500
Precipitation Buffer PTB Blue 2 1,700
Precipitation Star PS Yellow 6 1 star

Flow Cell Wash Kit (EXP-WSH004) contents

EXP-WSH004 kit contents v2

Contents Volume (µl) No. of tubes No. of uses
Wash Mix (WMX) 15 1 6
Wash Diluent (DIL) 1,300 2 6
Storage Buffer (S) 1,600 2 6
  • Wash Mix (WMX) contains DNase I.
  • Wash Diluent (DIL) contains the exonuclease buffer that maximises activity of the DNase I.
  • The Storage Buffer allows flow cells to be stored for extended periods of time.

To maximise the use of the Ultra-Long DNA Sequencing Kit V14, the EEB Expansion (EXP-EEB001) and the Ultra-Long Auxiliary Vials (EXP-ULA001) expansion packs are available.

These expansions provide extra library preparation and flow cell priming reagents to allow users to maximise the use out of their Ultra-Long DNA Sequencing Kit V14.

The EEB Expansion (EXP-EEB001) contains enough reagents for at least 6 standard extraction elution steps.

The Ultra-Long Auxiliary Vials (EXP-ULA001) provides enough reagents to carry out twelve additional flow cell loads on MinION or PromethION flow cells.

EEB Expansion (EXP-EEB001) contents:

EXP-EEB001 Kit content

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Extraction EB EEB White 1 6,000

Ultra-Long Auxiliary Vials (EXP-ULA001) contents:

EXP-ULA001 Kit content

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Elution Buffer EB Black 1 1,500
Sequencing Buffer UL SBU Red 2 1,000
Loading Solution UL LSU White cap, pink label 1 200
Flush Tether UL FTU Purple 1 600
Flow Cell Flush FCF Clear cap, light blue label 1 15,500

3. Isolation of white blood cells (WBCs) from whole blood

材料
  • 1.6 ml of whole blood
消耗品
  • RBC Lysis Solution (QIAGEN, 158106)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • 15 ml Falcon tubes
装置
  • Microfuge
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips

White blood cell sample preparation for the Ultra-long DNA experiment

Approximately 6 million isolated white blood cells must be prepared from 1.6 ml of whole blood to use as input in the Ultra-long DNA experiment.

Users may isolate white blood cells by any means they feel are most appropriate for the whole blood sample to be used. If 6 million cells have been isolated, users can start from the uHMW gDNA extraction step.

Add 4.8 ml of RBC Lysis Solution to 1.6 ml of whole blood in a 15 ml Falcon tube.

Gently invert the tube ten times to mix.

Incubate for 5 minutes at room temperature and gently invert twice during the incubation.

Centrifuge at 2000 x g for 2 minutes at 4°C to pellet the white blood cells.

Discard the supernatant by pouring. There will be ~200 µl supernatant remaining in the tube.

Resuspend the cells in the residual supernatant by gently flicking the tube.

Make up the volume to 1.6 ml with 1x PBS.

Repeat steps 1-7 twice more to complete three washes in total.

ヒント

If any red colouration persists, repeat the wash step until the cell pellet is white.

After the final spin, remove the entire supernatant by pouring and aspirating any remaining supernatant.

Resuspend the cell pellet in 40 µl 1x PBS. There will be approximately 6 million cells in the suspension.

最終ステップ

Take the cell pellet forward into the "uHMW gDNA extraction" step.

4. Preparation of tissue samples for gDNA extraction

材料
  • Cell Suspension Buffer (CSB): 0.35 M sucrose, 100 mM EDTA, 50 mM Tris.HCl pH 8
  • Frozen tissue sample
消耗品
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • 2.5 M sucrose
  • Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
  • 50 ml Falcon tubes
  • 5 ml Eppendorf DNA LoBind tubes
装置
  • Centrifuge suitable for 5 ml Eppendorf tubes (Eppendorf centrifuge 5804/5804 R or equivalent)
  • Eppendorf tube rack suitable for 5 ml Eppendorf tubes
  • Scalpel
  • TissueRuptor II (QIAGEN, cat # 9002755)
  • TissueRuptor Disposable Probes (QIAGEN, cat # 990890)
  • Florescent microscope with functionality to quantify nuclei (Logos CELENA S Digital Imaging System or equivalent)
  • Heat block equipped with thermoblock suitable for 5 ml Eppendorf tubes
  • 200 µm PluriStrainer® (pluriSelect, 43-50200-03)
  • 100 µm PluriStrainer® (pluriSelect, 43-50100-51)
  • 50 µm PluriStrainer® (pluriSelect, 43-50050-03)
  • 30 µm PluriStrainer® (pluriSelect, 43-50030-03)
  • PluriStrainer® Connector Ring (pluriSelect, 41-50000-03)
  • PluriStrainer® Funnel (pluriSelect, 42-50000)
  • P1000 pipette and tips
  • 10 ml syringe

Prepare the Cell Suspension Buffer (CSB) as follows:

Reagent Stock Final conc. Volume
Tris.HCl, pH 8 1 M 0.05 M 50 ml
EDTA 0.5 M 0.1 M 200 ml
Sucrose 2.5 M 0.35 M 140 ml
Nuclease-free water - - 610 ml
Total - - 1000 ml

Add 1 g of the frozen tissue sample to a weighing boat.

Using the scalpel, slice the tissue into thin strips and then dice the sample.

Transfer the tissue sample to a fresh 50 ml Falcon tube.

Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.

Using the QIAGEN TissueRuptor, gently homogenise the tissue sample.

  1. Insert the probe and pulse at minimum speed for one second. Stir the homogenate between each pulse.
  2. Repeat this five times.
重要

During homogenisation, only apply as much force as is required to gently break up the tissue. Excessive force will damage the nuclei and make them difficult to quantify. It is not a problem if there is intact material remaining at the end of this step, as it will be re-processed in later steps.

Assemble the pluriStrainer apparatus with a 200 μm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.

Pass the full volume of the tissue sample homogenate through the 200 µm PluriStrainer®.

The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.

Disassemble the pluriStrainer® apparatus according to the manufacturer's instructions, setting aside the strained homogenate in the 50 ml Falcon tube for later use.

Repeat the homogenisation process on any intact tissue caught by the pluriStrainer®:

  1. Transfer any intact tissue caught by the 200 µm pluriStrainer® into a fresh 50 ml Falcon tube by inverting the strainer and tapping out the intact tissue.
    Tip: A spatula can be used to help remove the intact tissue from the strainer.

  2. Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.

Repeat steps 6-10 two more times to perform a total of three rounds of tissue homogenisation.

Combine the contents of the 50 ml Falcon tube with the original strained homogenate set aside in step 10.

CHECKPOINT

The combined volume of 200 µm strained homogenate is ready for further processing.

Strain the 200 µm strained homogenate through the 100 µm pluriStrainer®:

  1. Assemble the pluriStrainer apparatus with a 100 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.

  2. Pass the full volume of the 200 µm strained homogenate through the 100 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.

  3. Disassemble the pluriStrainer® and retain the 100 µm strained homogenate in the 50 ml Falcon tube.

Strain the 100 µm strained homogenate through the 50 µm pluriStrainer®:

  1. Assemble the pluriStrainer apparatus with a 50 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.

  2. Pass the full volume of the 100 µm strained homogenate through the 50 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.

  3. Disassemble the pluriStrainer® and retain the 50 µm strained homogenate in the 50 ml Falcon tube.

Strain the 50 µm strained homogenate through the 30 µm pluriStrainer®:

  1. Assemble the pluriStrainer apparatus with a 30 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.

  2. Pass the full volume of the 50 µm strained homogenate through the 30 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.

  3. Disassemble the pluriStrainer® and retain the 30 µm strained homogenate in the 50 ml Falcon tube.

Determine the concentration of the nuclei in the purified homogenate using a fluorescent microscope and a stain appropriate for the nuclei in the sample.

Take forward a volume corresponding to 6 million nuclei and add this to a 5 ml Eppendorf DNA LoBind tube.

Centrifuge the 5 ml Eppendorf tube at 16,000 x g for five minutes to pellet the nuclei/cells.

Pipette off all the supernatant and discard, taking care not to disturb the pellet.

Add 40 µl of PBS to the 5 ml Eppendorf DNA LoBind tube.

Thoroughly mix the tube by repeatedly flicking. Ensure the pellet breaks up and no clumps remain in the nuclei/cell suspension.

Note: You may need to flick quite hard and thoroughly to ensure the pellet breaks up and no clumps remain.

最終ステップ

Take the nuclei/cell suspension forward into the "uHMW gDNA extraction" step.

5. uHMW gDNA extraction

材料
  • 6 million cells/nuclei isolated from frozen tissue or white blood cells isolated from whole blood
  • Extraction EB (EEB)
  • Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
消耗品
  • 5 ml Eppendorf DNA LoBind tubes
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • Isopropanol, 100% (Fisher, 10723124)
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • 2 ml Eppendorf DNA LoBind tubes
装置
  • Heat block set at 56°C
  • Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
  • Hula mixer（緩やかに回転するミキサー）
  • Microfuge
  • P1000 pipette and wide-bore pipette tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
  • Eppendorf 5424 centrifuge (or equivalent)

This method does NOT use the Monarch Elution Buffer II from the Monarch® HMW DNA Extraction Kit.

This method has been optimised using the Extraction EB (EEB) from the Oxford Nanopore sequencing kit.

重要

Ensure ethanol is added to the Monarch gDNA Wash Buffer as per kit guidance.

Thaw the Extraction EB (EEB) at room temperature, mix by vortexing and place on ice.

Add 6 million cells resuspended in 40 µl PBS to a fresh 5 ml tube. Cells can be isolated from cell culture, white blood cells from blood, or tissue according to the above methods.

ヒント

Thorough but gentle resuspension of cells is required to ensure efficient lysis and to prevent heterogeneity in the subsequent steps.

In a separate 2 ml Eppendorf DNA LoBind tube, combine the following reagents:

Reagent Volume
Monarch HMW gDNA Tissue Lysis Buffer 1,800 µl
Proteinase K 60 µl
Total 1860 µl

Add 1.8 ml of mixed Monarch HMW gDNA Tissue Lysis Buffer and Proteinase K to the resuspended cells.

Gently mix by slowly pipetting the reaction five times using a 1 ml wide-bore pipette tip.

ヒント

When using cell lines, we have found that the incubation step below can be omitted.

Incubate the reaction at 56°C for 10 minutes.

Using a regular pipette tip, add 15 µl of Monarch RNase A.

Gently mix by slowly pipetting the reaction five times using a 1 ml wide-bore pipette tip.

Incubate the reaction at 56°C for 10 minutes on a thermomixer at 650 rpm.

ヒント

When using cell lines, we have found the protein removal steps can be omitted. If using cell lines, proceed directly to step 13.

Using a regular pipette tip, add 900 µl of the Monarch Protein Separation Solution to the reaction and mix using a Hula Mixer (rotator mixer) for 10 minutes, rotating at 3 rpm.

Centrifuge the reaction at 16,000 x g for 10 minutes at 4°C to separate the protein from the DNA.

DNA will be present in the upper phase, whereas protein and other contaminants will be in the lower phase.

Using a wide-bore pipette tip, carefully aspirate the upper phase containing the DNA and transfer to a fresh 5 ml tube without disturbing the phase below.

The DNA in the upper phase should be extremely viscous and should only be possible to aspirate using a wide-bore pipette tip.

ヒント

If the protein phase is disturbed, the tube can be centrifuged again at 16,000 x g for 10 minutes at 4°C.

Add three Monarch DNA Capture Beads to the collected DNA phase (or to the lysis reaction if proceeded directly from Step 9).

Note: The first bead is a sacrificial bead and will remain at the bottom of the tube throughout the remainder of the process.

Add 2.5 ml isopropanol to the tube and mix using a Hula Mixer (rotator mixer) for 20 minutes rotating at 3 rpm. Ensure the DNA has fully precipitated around the glass beads.

ヒント

Check the DNA is binding to the beads by looking for a viscous mass around the beads. The mixing step can be extended if the DNA is not obviously condensing around the beads.

Leave the tube to stand for 1 minute, without rotating, at room temperature.

Aspirate the supernatant from the tube, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any supernatant remaining in the lid of the tube.

Note: if ~100 µl of supernatant is remaining in the tube, perfomance will not be affected.

Add 2 ml of Monarch gDNA Wash Buffer to the tube containing DNA bound to the beads and invert the tube to mix.

Ensure ethanol is added to the Monarch gDNA Wash Buffer as per kit guidance.

Aspirate the Wash Buffer, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any Wash Buffer remaining in the lid of the tube.

Add 2 ml of Monarch gDNA Wash Buffer to the tube containing the DNA bound to the beads.

To a fresh 2 ml Eppendorf tube, add 560 µl of Extraction EB (EEB).

Aspirate the Wash Buffer, being careful not to aspirate the DNA that is bound to the beads. Check for and remove any Wash Buffer remaining in the lid of the tube.

Transfer the beads to a Monarch Bead Retainer inserted in a Monarch Collection Tube II.

Briefly spin the tube using a microfuge to remove any remaining Wash Buffer from the beads. Dispose of the collection tube containing residual wash buffer.

重要

Do NOT use the Monarch Elution Buffer II in the Monarch® HMW DNA Extraction Kit for Tissue.

Immediately transfer the beads from the bead retainer into the 2 ml tube containing 560 µl of Extraction EB (EEB).

重要

Beads should be transferred immediately to ensure that they do not over-dry, which could lead to increased solubilisation times.

Incubate the tube for 10 minutes at 56°C.

Pour the eluate and beads into a clean bead retainer inserted in a collection tube. Spin the tube at 1000 x g for 1 minute to separate eluate from the beads. Dispose of beads and bead retainer.

Add 200 µl of Extraction EB (EEB) to the collection tube to bring the total elution volume to 760 µl.

Transfer the eluate to a fresh 2 ml Eppendorf DNA LoBind tube.

Incubate the eluate for 10 minutes at 56°C.

Gently mix the eluate by slowly pipetting 10 times using a 1 ml wide-bore pipette tip.

Thorough but gentle resuspension of DNA is required to prevent heterogeneity in the sample.

最終ステップ

Take forward the resuspended DNA into the quantification step. However, at this point it is possible to store the sample at room temperature overnight.

6. (Optional) gDNA quantification

材料
  • Monarch® DNA Capture Beads
  • Monarch® Bead Retainer
  • Monarch® Collection Tubes II
消耗品
  • 2 ml Eppendorf DNA LoBind tubes
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
装置
  • ボルテックスミキサー
  • Centrifuge
  • Qubit fluorometer (or equivalent)
  • P200 pipette and tips

Quantification of uHMW gDNA

The method to quantify uHMW gDNA was developed by Paul A ‘Giron’ Koetsier & Eric J Cantor, 2021, which recommends the use of a regular P200 pipette and tip.

This optional uHMW gDNA quantification step has also been included in the protocol for user QC. However, this step can be omitted and 750 µl of DNA in Extraction EB (EEB) can be taken straight into the tagmentation step of the protocol.

Use a regular P200 pipette tip to aspirate 10 µl of gDNA.

ヒント

If the DNA is particularly viscous, the aspirated DNA can be separated from the sample by forcing the sample against the side of the tube to break the DNA off. It is critical that the DNA is completely homogenous, so that the 10 µl of sample that is removed is representative of the entire sample.

Dispense the aspirated gDNA into a fresh 2 ml Eppendorf DNA LoBind tube.

Add a Monarch DNA Capture Bead to the 10 µl of gDNA and vortex aggressively for 1 minute to shear the gDNA.

Transfer the gDNA and beads into a clean Monarch Bead Retainer inserted in a Monarch Collection Tube II. Spin the tube at 1000 x g for 1 minute to separate gDNA from the beads. Dispose of beads and bead retainer.

Using a wide-bore pipette tip, transfer the gDNA into a clean Eppendorf DNA LoBind tube.

The size of Eppendorf DNA LoBind tube depends on if you are using the Precipitation Star (PS) during the clean-up step:

Clean-up using the Precipitation Star (PS)

  • Transfer the gDNA into a clean 1.5 ml Eppendorf DNA LoBind tube.

Clean-up not using the Precipitation Star (PS):

  • Transfer the gDNA into a clean 2 ml Eppendorf DNA LoBind tube.
CHECKPOINT

Quantify the sample using a Qubit fluorometer. The expected yield is 30-40 µg of DNA.

最終ステップ

Take forward 750 µl DNA into the tagmentation step.

7. Tagmentation

材料
  • 750 µl of extracted uHMW gDNA in EEB
  • Rapid Adapter (RA)
  • Fragmentation Mix (FRA)
  • FRA Dilution Buffer (FDB)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
装置
  • Thermal cycler or heat block
  • Microfuge
  • P1000 pipette and wide-bore pipette tips
  • P1000 pipette and tips
  • P20 ピペットとチップ
  • Ice bucket with ice

Best practice for handling uHMW gDNA

When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.

To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.

While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.

For further information, please refer to the troubleshooting section.

Thaw the the kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:

Once thawed, keep all the kit components on ice.

Reagent Thaw at room temperature Briefly spin down Mix well by pipetting
Fragmentation Mix (FRA) Not frozen ✓ ✓
FRA dilution buffer (FDB) Not frozen ✓ ✓
Rapid Adapter (RA) Not frozen ✓ ✓

In a 1.5 ml Eppendorf DNA LoBind tube, dilute the Fragmentation Mix (FRA) with FRA Dilution Buffer (FDB) as follows:

Reagent Volume
Fragmentation Mix (FRA) 6 µl
FRA dilution buffer (FDB) 244 µl
Total 250 µl

Mix the diluted Fragmentation Mix (FRA) by pipetting.

Using a regular pipette tip, add 250 µl of diluted Fragmentation Mix (FRA) to the 750 µl of extracted DNA. Stir the reaction with the pipette tip whilst expelling the diluted Fragmentation Mix (FRA) to ensure an even distribution.

Immediately mix the reaction by slowly pipetting 10 times with a wide-bore pipette tip.

Visually check the reagents are thoroughly mixed. It is important to immediately mix the diluted Fragmentation Mix (FRA) with the DNA thoroughly.

Incubate the reaction as follows:

Temperature Time
Room temperature 10 minutes
75°C 10 minutes
On ice Cool on ice for a minimum of 10 minutes

Note: the reaction must be cooled on ice before adding Rapid Adapter (RA) to prevent denaturing the enzyme.

Add 5 µl Rapid Adapter (RA) to the reaction using a regular pipette tip.

Gently mix the reaction by slowly pipetting five times using a 1 ml wide-bore pipette tip.

Note: visually check to ensure the reaction is thoroughly mixed.

Incubate the reaction for 30 minutes at room temperature.

8. Clean-up (using the precipitation star)

材料
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Precipitation Buffer (PTB)
  • Precipitation Star (PS)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
装置
  • Centrifuge
  • Microfuge
  • Hula mixer（緩やかに回転するミキサー）
  • P200 pipette and tips
  • P1000 pipette and tips
  • P1000 pipette and wide-bore pipette tips
重要

Ensure you are using the correct method depending on your batch of Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114).

We had previously communicated an issue with the Precipitation Star (PS) found in the SQK-ULK114 kits and updated the protocol to address it.

We have since improved our manufacturing and internal validation processes and are now in the position to reintroduce the use of the Precipitation Star (PS) within the SQK-ULK114 protocol.

Kit format where stars should not be used: Kit format with improved precipitation stars that can be used:
Batch ULK114.20.xxxx or older
ULK114 Kit format where stars should not be used		 Batch ULK114.30.0001 or newer
ULK114 Kit format with improved Precipitation stars

Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by vortexing as indicated by the table below:

Reagent Thaw at room temperature Briefly spin down Mix well by pipetting
Precipitation buffer (PTB) ✓ ✓ ✓
Elution Buffer (EB) ✓ ✓ ✓

Once thawed, keep all the kit components on ice.

ヒント

We strongly recommend using a 1.5 ml Eppendorf DNA LoBind tube for the clean-up method when using the Precipitation Star (PS)

Add a Precipitation Star (PS) to the sample.

Using a regular pipette tip, add 500 µl of Precipitation Buffer (PTB) to the sample.

Mix the sample by rotating on a Hula Mixer (rotator mixer) for 20 minutes at 3 rpm.

Visually inspect to check the DNA has precipitated around the Precipitation Star (PS).

Using a regular pipette tip, carefully remove the supernatant from the tube, taking care not to aspirate the DNA.

In the 1.5 ml Eppendorf LoBind DNA tube, the Precipitation Star (PS) should be suspended mid-way in the tube, allowing the supernatant beneath the star to be removed.

Briefly spin down the tube and remove any residual supernatant using a regular pipette tip, taking care not to aspirate the DNA.

Using a regular pipette tip, add 300 µl of Elution Buffer (EB) to the tube containing the Precipitation Star (PS) and DNA. Incubate overnight at room temperature, for a minimum of 12 hours.

Using a wide-bore pipette tip, remove and retain eluate containing the DNA library into a clean 1.5 ml Eppendorf LoBind DNA tube.

Briefly spin down the tube containing the Precipitation Star (PS) and remove any remaining eluate with a wide-bore pipette tip, ensuring there is no liquid remaining on the Precipitation Star (PS).

Dispose of the tube containing the Precipitation Star (PS).

Gently mix the DNA library by slowly pipetting 10 times with a wide-bore pipette tip.

Thorough but gentle resuspension of DNA is required to prevent heterogeneity in the sample.

最終ステップ

Take the DNA library forwards for loading into the flow cell. Store the library on ice until ready to load.

ヒント

推奨のライブラリー保存方法

短期間の保存や繰り返し使用する場合は__（例　フローセルをウオッシュして再度ロードする場合）は、ライブラリーをEppendorf DNA LoBindチューブに入れ、__4℃で保存 することをお勧めします。 __3か月以上の長期保存の場合は、____ライブラリーをEppendorf DNA LoBindチューブに -80 ° Cで保存 することをお勧めします。

9. Clean-up (no precipitation star)

材料
  • Elution Buffer from the Oxford Nanopore kit (EB)
  • Precipitation Buffer (PTB)
消耗品
  • 2.0 ml Eppendorf DNA LoBind tubes
装置
  • Centrifuge
  • Microfuge
  • Hula mixer（緩やかに回転するミキサー）
  • P200 pipette and tips
  • P1000 pipette and tips
  • P1000 pipette and wide-bore pipette tips
重要

Ensure you are using the correct method depending on your batch of Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114).

We had previously communicated an issue with the Precipitation Star (PS) found in the SQK-ULK114 kits and updated the protocol to address it.

We have since improved our manufacturing and internal validation processes and are now in the position to reintroduce the use of the Precipitation Star (PS) within the SQK-ULK114 protocol.

Kit format where stars should not be used: Kit format with improved precipitation stars that can be used:
Batch ULK114.20.xxxx or older
ULK114 Kit format where stars should not be used		 Batch ULK114.30.0001 or newer
ULK114 Kit format with improved Precipitation stars

Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by vortexing as indicated by the table below:

Reagent Thaw at room temperature Briefly spin down Mix well by pipetting
Precipitation buffer (PTB) ✓ ✓ ✓
Elution Buffer (EB) ✓ ✓ ✓

Once thawed, keep all the kit components on ice.

ヒント

We recommend using a 2 ml Eppendorf DNA LoBind tube for the clean-up method when not using the Precipitation Star (PS).

Using a regular pipette tip, add 500 µl of Precipitation Buffer (PTB) to the sample.

Mix the sample by rotating on a Hula Mixer (rotator mixer) for 20 minutes at 3 rpm.

Visually inspect to check the DNA has precipitated, forming a glassy white mass.

Centrifuge the sample at 1000 x g for 1 minute.

Using a regular pipette tip, carefully remove the supernatant from the tube, taking care not to aspirate the DNA pellet.

Centrifuge the sample at 1000 x g for 1 minute.

Using a regular pipette tip, carefully remove any residual supernatant from the tube, taking care not to aspirate the DNA pellet.

Using a regular pipette tip, add 300 µl of Elution Buffer (EB) to the tube containing the DNA. Incubate overnight at room temperature, for a minimum of 12 hours.

Gently mix the DNA library by slowly pipetting 10 times with a wide-bore pipette tip.

Thorough but gentle resuspension of DNA is required to prevent heterogeneity in the sample.

最終ステップ

Take the DNA library forwards for loading into the flow cell. Store the library on ice until ready to load.

ヒント

推奨のライブラリー保存方法

短期間の保存や繰り返し使用する場合は__（例　フローセルをウオッシュして再度ロードする場合）は、ライブラリーをEppendorf DNA LoBindチューブに入れ、__4℃で保存 することをお勧めします。 __3か月以上の長期保存の場合は、____ライブラリーをEppendorf DNA LoBindチューブに -80 ° Cで保存 することをお勧めします。

10. Priming and loading the SpotON flow cell

材料
  • Flow Cell Flush (FCF)
  • Flush Tether UL (FTU)
  • Loading Solution UL (LSU)
  • Sequencing Buffer UL (SBU)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • MinIONとGridIONのFlow Cell
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
装置
  • MinIONかGridION のデバイス
  • MinIONとGridIONのFlow Cell ライトシールド
  • P1000 pipette and wide-bore pipette tips
  • P200 pipette and wide-bore pipette tips
  • P1000 ピペット及びチップ
  • P200 ピペットとチップ
  • P20 ピペットとチップ
重要

Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).

Only use the reagents provided with the SQK-ULK114 kit for priming and loading the flow cell. Reagents from other kits are not compatible with this protocol.

ヒント

フローセルのプライミングとローディング

新規ユーザーは、 初回使用前に'Priming and loading your flow cell' のビデオをご覧いただくことをお勧めします。

重要

Ensure the MinION Flow Cell Light Shield is not installed on to your flow cell until after loading your DNA library.

To ensure optimal interactions and access to the flow cell ports, please ensure the MinION Flow Cell Light Shield is not installed on your flow cell until after loading your DNA library.

If the MinION Flow Cell Light Shield has already been installed, remove it from the flow cell and store until required later in the protocol.

Thaw the Sequencing Buffer UL (SBU), Loading Solution UL (LSU), Flush Tether UL (FTU) and one tube of Flow Cell Flush (FCF) at room temperature and mix by vortexing. Then spin down and place on ice.

In a new tube, prepare the DNA library for loading as follows. Use a wide-bore pipette tip for the addition of the DNA library:

Reagent Volume per flow cell
Sequencing Buffer UL (SBU) 37.5 µl
Loading Solution UL (LSU) 3.7 µl
DNA library 33.8 µl
Total 75 µl

Note: Ensure the Sequencing Buffer UL (SBU) and Loading Solution UL (LSU) are thoroughly mixed by pipetting before the addition of the DNA library.

Gently mix the prepared DNA library by slowly pipetting ten times using a wide-bore pipette tip.

Incubate at room temperature for 30 minutes then gently mix by slowly pipetting with a wide-bore tip. Visually inspect to ensure the sample is homogenous.

MinIONまたはGridIONデバイスの蓋を開け、フローセルをクリップの下にスライドさせます。 フローセルをしっかりと押さえ、サーマルプレートと電気接触が密着しているかを確認してください。

Flow Cell Loading Diagrams Step 1a_JP

Flow Cell Loading Diagrams Step 1b_JP

オプショナルステップ

ライブラリーをロードする前にフローセルチェックを行い、使用可能なポアの数を把握して下さい。

フローセルが以前にチェックされている場合は、このステップを省略できます。

詳細については、MinKNOWプロトコルのフローセルチェックの手順 flow cell check instructionsを参照してください。

フローセルのプライミングポートカバーを時計方向にスライドさせ、プライミングポートを開きます。

Flow Cell Loading Diagrams Step 2_JP

重要

フローセルからバッファーを引き上げる際には注意してください。20～30μl以上は除去せず、ポアのアレイ全体が常にバッファーで覆われていることを確認して下さい。アレイに気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。

プライミングポートを開けた後に、カバーの下に小さな気泡がないかを確認して下さい。気泡を取り除くために少量の液を引き上げます。

  1. P1000ピペットを200 µ Lに設定して下さい。
  2. ピペットの先端をプライミングポートに差し込みます。
  3. 目盛りが220-230 ulと表示されるまでダイヤルを回して、20-30 ulを吸い上げるか、少量のバッファーがピペットの先端に入るのが見えるまでダイヤルを回します。

(注： プライミングポートからセンサーアレイ全体にバッファーがあることを確認してください。

Flow Cell Loading Diagrams Step 03 V5_JP

重要

For optimal sequencing performance and improved output on R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml. We do not recommend using recombinant BSA.

For optimal sequencing performance and improved output on R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml. We do not recommend using recombinant BSA.

To prepare the flow cell priming mix with BSA, combine the following reagents in a 1.5 ml Eppendorf tube, and mix by inverting the tube and pipette mix at room temperature:

Reagent Volume
Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl
Flush Tether UL (FTU) 30 µl
Flow Cell Flush (FCF) 1170 µl
Total 1205 µl
重要

Ensure the MinION Flow Cell Light Shield is not installed on the flow cell at this stage.

If the MinION Flow Cell Light Shield has already been installed, remove it from the flow cell and store until required later in the protocol.

Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes.

Flow Cell Loading Diagrams Step 04 V5

フローセルのプライミングを完了させます。

  1. SpotON サンプルポートカバーをゆっくりと持ち上げ、SpotON サンプルポートにアクセスできるようにします。
  2. 200μlのプライミングミックスをフローセルのプライミングポート（SpotONサンプルポートではありません）に気泡が入らないように注入します。

Flow Cell Loading Diagrams Step 5_JP

Flow Cell Loading Diagrams Step 06 V5_JP

Ensure the SpotON port and Priming port covers of the flow cell are open in preparation for loading.

GridION Step 1

Using a wide-bore pipette tip, load the DNA library onto the SpotON port until 75 μl has been loaded.

Take care not to place the pipette tip directly onto/into the SpotON port as this could damage the array.

Allow the DNA library to flow through the SpotON port by waiting up to two minutes.

If the DNA library does not enter the SpotON port, apply negative pressure in the flow cell as explained further below.

GridION ULK lib load

Cover Waste port 2 and the Priming port with clean, gloved fingers.

GridION Step 4

Using a fully depressed P200 pipette, insert the tip in Waste port 1 whilst Waste port 2 and the Priming port are covered.

GridION Step 5 6

Very slowly aspirate to pull the DNA library into the SpotON sample port. Closely watch the DNA library on the SportON port and completely remove the pipette as soon as the library starts to be pulled into the port.

Note: Take care to not apply too much negative pressure too quickly to avoid bringing air bubbles into the flow cell. Air bubbles will cause irreversible damage to the flow cell.

GridION-Step 7 8 9 looping

SpotONサンプルポートカバーをゆっくりと元に戻し、バング（カバーの先）がSpotONポートに入ることを確認し、プライミングポートを閉じます。

Step 8 update_JP

Flow Cell Loading Diagrams Step 9_JP

重要

最適なシークエンス出力を得るために、ライブラリーがロードされたすぐにライトシールドをフローセルに取り付けてください。

ライブラリーがフローセル上にある状態では（ウォッシングやリロードのステップを含める）、フローセルにライトシールドを付けたままにしておくことを推奨します。ライトシールドは、ライブラリーがフローセルから除去された時点で取り外すことができます。

ライトシールドを以下のようにフローセルに設置して下さい。

  1. ライトシールドの先端を慎重にクリップに当てます。 (注： ライトシールドをクリップの下に無理に押し込まないでください。

  2. ライトシールドをフローセルにゆっくりと下ろします。ライトシールドは、フローセルの上部全体を覆うようにSpotONカバーの周囲に取り付けます。

J2264 - Light shield animation Flow Cell FAW optimised-Japanese step10

注意

MinIONフローセルライトシールドは、フローセルに固定されていないため、取り付け後の取り扱いには注意が必要です。

最終ステップ

デバイスの蓋を閉め、MinKNOWでシークエンスランをセットします。

We recommend loading an ultra-long DNA library three times per flow cell to increase output.

A nuclease wash using the Flow Cell Wash Kit (EXP-WSH004) is required between each subsequent library load to recover channels and maximise sequencing output.

For MinION/GridION flow cells, there is enough library generated for six consecutive loads per reaction, using 33.8 µl of fresh library combined with 37.5 µl of Sequencing Buffer (SBU) and 3.7 µl of Loading Solution (LSU) before re-loading for further sequencing.

Please follow Flushing a MinION/GridION Flow Cell in the Flow Cell Wash Kit protocol for the nuclease wash instructions. To run another library straight away, follow the instructions below.

11. Reloading ultra-long DNA library on a MinION/GridION flow cell

材料
  • Flow Cell Wash Kit (EXP-WSH004)
  • Flush Tether UL (FTU)
  • Flow Cell Flush (FCF)
  • Sequencing Buffer UL (SBU)
  • Loading Solution UL (LSU)
消耗品
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Bovine Serum Albumin (BSA) (50 mg/ml) (e.g Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)
装置
  • P1000 pipette and wide-bore pipette tips
  • P200 pipette and wide-bore pipette tips
  • P1000 pipette and tips
  • P200 ピペットとチップ
  • P20 ピペットとチップ
重要

Before reloading your library or loading a new library, please ensure you wash the flow cell using the Flow Cell Wash Kit (EXP-WSH004).

Follow the instructions in the Flow Cell Wash Kit (EXP-WSH004) for MinION/GridION protocol.

  • This washing procedure aims to remove most of the initial library and prepare the flow cell for loading of a subsequent library.
  • Data acquisition in MinKNOW should be paused during the wash procedure and library loading.
  • After the flow cell has been washed, another library can be loaded.
ヒント

We recommend keeping the light shield on the flow cell during washing if a second library will be loaded straight away.

If the flow cell is to be washed and stored, the light shield can be removed.

To run a second library of ultra-long DNA straight after flushing a flow cell, we recommend removing all fluid from the waste channel after each priming step.

Thaw the Sequencing Buffer UL (SBU), Loading Solution UL (LSU), Flush Tether UL (FTU) and one tube of Flow Cell Flush (FCF) at room temperature and mix by vortexing. Then spin down and place on ice.

In a new tube, prepare the DNA library for loading as follows. Use a wide-bore pipette tip for the addition of the DNA library:

Reagent Volume per flow cell
Sequencing Buffer UL (SBU) 37.5 µl
Loading Solution UL (LSU) 3.7 µl
DNA library 33.8 µl
Total 75 µl

Note: Ensure the Sequencing Buffer UL (SBU) and Loading Solution UL (LSU) are thoroughly mixed by pipetting before the addition of the DNA library.

Gently mix the prepared DNA library by slowly pipetting ten times using a wide-bore pipette tip.

Incubate at room temperature for 30 minutes then gently mix by slowly pipetting with a wide-bore tip. Visually inspect to ensure the sample is homogenous.

Slide the priming port cover of the flow cell clockwise to open the priming port.

Flow Cell Loading Diagrams Step 2

重要

フローセルからバッファーを引き上げる際には注意してください。20～30μl以上は除去せず、ポアのアレイ全体が常にバッファーで覆われていることを確認して下さい。アレイに気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。

プライミングポートを開けた後に、カバーの下に小さな気泡がないかを確認して下さい。気泡を取り除くために少量の液を引き上げます。

  1. P1000ピペットを200 µ Lに設定して下さい。
  2. ピペットの先端をプライミングポートに差し込みます。
  3. 目盛りが220-230 ulと表示されるまでダイヤルを回して、20-30 ulを吸い上げるか、少量のバッファーがピペットの先端に入るのが見えるまでダイヤルを回します。

(注： プライミングポートからセンサーアレイ全体にバッファーがあることを確認してください。

Flow Cell Loading Diagrams Step 03 V5_JP

重要

MinION R10.4.1フローセル（FLO-MIN114）での最適なシークエンス性能と出力向上のために、フローセルのプライミングミックスに最終濃度0.2 mg/mlでBovine Serum Albumin (BSA) を添加することを推奨します。

(注： その他のアルブミンの種類（組換えヒト血清アルブミンなど）の使用は推奨しません。

To prepare the flow cell priming mix with BSA, combine the following reagents in a 1.5 ml Eppendorf tube, and mix by inverting the tube and pipette mix at room temperature:

Reagent Volume
Bovine Serum Albumin (BSA) at 50 mg/ml 5 µl
Flush Tether UL (FTU) 30 µl
Flow Cell Flush (FCF) 1170 µl
Total 1205 µl

Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes.

Flow Cell Loading Diagrams Step 04 V5

重要

It is vital to wait five minutes between the priming mix flushes to ensure effective removal of the nuclease.

Close the priming port cover and ensure the SpotON sample port cover is closed.

Flow Cell Loading Diagrams Step 9

重要

It is vital that the flow cell priming port and SpotON sample port are closed before removing the waste buffer to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Remove the waste buffer, as follows:

  1. Close the priming port and SpotON sample port cover, as indicated in the figure below.
  2. Insert a P1000 pipette into waste port 1 and remove the waste buffer.

Note: As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

Flow cell ports

Slide open the priming port and load 200 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.

Close the priming port and use a P1000 to remove all fluid from the waste channel through Waste Port 1.

Ensure the SpotON port and Priming port covers of the flow cell are open in preparation for loading.

GridION Step 1

Using a wide-bore pipette tip, load the DNA library onto the SpotON port until 75 μl has been loaded.

Take care not to place the pipette tip directly onto/into the SpotON port as this could damage the array.

Allow the DNA library to flow through the SpotON port by waiting up to two minutes.

If the DNA library does not enter the SpotON port, apply negative pressure in the flow cell as explained further below.

GridION ULK lib load

Cover Waste port 2 and the Priming port with clean, gloved fingers.

MinION Mk1B Step 4

Using a fully depressed P200 pipette, insert the tip in Waste port 1 whilst Waste port 2 and the Priming port are covered.

MinION Mk1B Step 5 6

Very slowly aspirate to pull the DNA library into the SpotON sample port. Closely watch the DNA library on the SportON port and completely remove the pipette as soon as the library starts to be pulled into the port.

Note: Take care to not apply too much negative pressure too quickly to avoid bringing air bubbles into the flow cell. Air bubbles will cause irreversible damage to the flow cell.

Mk1B-Step 7 8 9 looping

SpotONサンプルポートカバーをゆっくりと元に戻し、バング（カバーの先）がSpotONポートに入ることを確認し、プライミングポートを閉じます。

Step 8 update_JP

Flow Cell Loading Diagrams Step 9_JP

重要

最適なシークエンス出力を得るために、ライブラリーがロードされたすぐにライトシールドをフローセルに取り付けてください。

ライブラリーがフローセル上にある状態では（ウォッシングやリロードのステップを含める）、フローセルにライトシールドを付けたままにしておくことを推奨します。ライトシールドは、ライブラリーがフローセルから除去された時点で取り外すことができます。

ライトシールドを以下のようにフローセルに設置して下さい。

  1. ライトシールドの先端を慎重にクリップに当てます。 (注： ライトシールドをクリップの下に無理に押し込まないでください。

  2. ライトシールドをフローセルにゆっくりと下ろします。ライトシールドは、フローセルの上部全体を覆うようにSpotONカバーの周囲に取り付けます。

J2264 - Light shield animation Flow Cell FAW optimised-Japanese step10

注意

MinIONフローセルライトシールドは、フローセルに固定されていないため、取り付け後の取り扱いには注意が必要です。

Once the flow cell is reloaded, resume the sequencing run on MinKNOW and trigger a pore scan.

To resume sequencing run, navigate to the Experiments page, click 'Resume' and select flow cell position.

To manually trigger a channel scan, click 'Start pore scan' and select flow cell position.

For further information, please see the MinKNOW protocol.

Resume run:

Resume Run ULK114 1

Resume Run ULK114 2 FC select

Pore scan:

Start pore scan ULK 1

Start pore scan ULK 2 FC select

12. Data acquisition and basecalling

シークエンスの開始方法

フローセルをロードしたら、MinKNOWでシークエンシングランを開始できます。MinKNOWは、装置、データ収集、リアルタイムベースコールを制御するシークエンスソフトウェアです。 MinKNOWの設定と使用の詳細については、MinKNOW Protocolを参照してください。

MinKNOWは、複数の方法でシークエンスの設定をすることができます。

  • シークエンシングデバイスに直接かリモートで接続されているコンピューター。
  • GridION、Minion Mk1C、またはPromethion 24/48シークエンシングデバイスで直接使用できます。

シークエンス装置でMinKNOWを使用する方法の詳細については、装置のユーザーマニュアルを参照してください。

MinKNOWでシークエンスランを開始するには、次の手順に従います。

1. スタートページに移動し、 Start sequencing をクリックします。

2. 名前、フローセルの位置、サンプルIDなどの実験の詳細を入力します。

3. キットのページで、ライブラリー調製に使用するシークエンシングキットを選択します。

4. シークエンスランのパラメータの変更を設定（ランオプションの変更など）するか、デフォルト設定のままにします。

(注： シークエンスランの設定時にベースコールがオフになっていた場合には、MinKNOWでポストラン・ベースコールを後日に実行することも出来ます。詳細については、MinKNOW protocolを参照してください。

5. Start をクリックしてシークエンスランを開始します。

シークエンシング後のデータ解析

MinKNOWでシークエンスが終わると、フローセルを再利用または返却ができます。詳しくは、フローセルの再利用と返却のセクションをご覧ください。

シークエンシングとベースコールの後にはデータを解析することができます。 ベースコールおよびベースコール後の解析オプションの詳細については、Data Analysis を参照してください。

ダウンストリーム解析セクションでは、データを解析するためのオプションの概要を説明しています。

13. フローセルの再利用と返却

材料
  • Flow Cell Wash Kit (EXP-WSH004)

シークエンス実験終了後、フローセルを再利用する場合は、Flow Cell Wash Kitのプロトコールに従い、洗浄したフローセルを2～8℃で保管してください。

Flow Cell Wash Kit protocolは、Nanoporeコミュニティーで入手できます。

ヒント

運転を停止したらできるだけ早くフローセルをウォッシュすることをお勧めします。しかし、これが不可能な場合はフローセルをデバイスに入れたまま、翌日にウォッシュをして下さい。

または、返送手順に従って、オックスフォード・ナノポアに返送してください。

フローセルの返却方法は hereをご覧ください。

(注： 製品を返却する前に、すべてのフローセルを脱イオン水で洗浄する必要があります。

重要

シークエンシング実験に関して問題が発生した場合や質問がある場合には、このプロトコルのオンライン版にあるトラブルシューティングガイドを参照してください。

14. Downstream analysis

Post-basecalling analysis

There are several options for further analysing your basecalled data:

1. EPI2ME workflows

For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, please refer to the resource centre and search for bioinformatics tools for your application. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

15. Issues during library preparation

以下は、最もよく起こる問題のリストであり、いくつかの原因と解決策が提案されています。

Nanopore Community Support セクションにFAQをご用意しています。

ご提案された解決策を試しても問題が解決しない場合は、テクニカルサポートに電子メール (support@nanoporetech.com)または LiveChat in the Nanopore Communityでご連絡ください。

Troubleshooting

Observation Comments and actions
Low throughput 1. Vortex gently after adding the diluted Fragmentation Mix (FRA) to break up the largest fragments.
2. Ensure the diluted Fragmentation Mix (FRA) is thoroughly mixed with the gDNA.
3. Use less input material if the DNA library was too viscous to load onto the flow cell.
DNA is too viscous and will not load onto a flow cell 1. Lower the input material to reduce the amount of gDNA going into the library preparation and reduce viscosity.
2. If DNA library will not load using the method outlined in this protocol, slowly pipette mix 5 times with a standard P200 pipette set to the full volume of the library and reload the flow cell.
Read lengths are not long enough 1. Increase input material.
Note: Library viscosity increases with more gDNA.
2. Reduce volume of Fragmentation Mix (FRA) added to FRA Dilution Buffer (FDB) to avoid over-fragmentation of gDNA.
Note: We do not recommend diluting less than 2 µl Fragmentation Mix (FRA).
3. We recommend using PFGE to check the extracted gDNA is of ultra-high molecular weight (uHMW), thus capable of generating ultra-long read lengths.
No sequencing output 1. Check gDNA has been recovered in library preparation using a NanoDrop spectrophotometer.
2. Check viscosity of the sample. The library should be viscous if it contains uHMW gDNA in this protocol.
Aspirating supernatant when the DNA has precipitated Take care to not aspirate the DNA. Remove smaller volumes of supernatant incrementally to reduce the risk of aspirating the DNA.
Mixing Mix slowly and carefully to prevent DNA shearing. Low vortexing can be used to mix at the expense of ultra-long reads. With vortexing, long read lengths of ~90 kb N50 can still be generated with improved outputs.
No DNA recovered from the library preparation clean-up If the DNA is no longer viscous or the NanoDrop reading is low, DNA may have been lost during the clean-up step of the library preparation.
1. Ensure uHMW DNA is used or users risk DNA loss.
2. Take care to not aspirate the precipitated DNA during the clean-up step. To avoid this, remove smaller volumes of supernatant incrementally. Ensure as much supernatant is removed as possible.

Last updated: 12/11/2024

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