Protocol

Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) VULK_9177_v114_revM_27Nov2022

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

Overview

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_revM_27Nov2022

1. Overview of the protocol

IMPORTANTE

Este es un producto de acceso anticipado

Para tener más información sobre los programas de acceso anticipado, consulte este artículo sobre las fases de lanzamiento del producto.

Procure usar la versión más reciente del protocolo.

Ultra-Long DNA Sequencing Kit features:

This kit is recommended for users who:

Want to reliably generate ultra-long read length N50s (>50 kb), with yields of 10-20+ Gbases on MinION/GridION
Sequence long reads from extracted uHMW DNA

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

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.

IMPORTANTE

Compatibility of this protocol

This protocol should only be used in combination with:

Ultra-Long DNA Sequencing Kit (SQK-ULK114)
Flow Cell Wash Kit (EXP-WSH004)
R10.4.1 flow cells (FLO-MIN114)
EEB Expansion (EXP-EEB001)
Ultra-Long Auxiliary Vials (EXP-ULA001)

2. Equipment and consumables

Material

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) (kit de lavado de celda de flujo)

Consumibles

1.5 ml Eppendorf DNA LoBind tubes
2 ml Eppendorf DNA LoBind tubes
5 ml Eppendorf DNA LoBind tubes
15 ml Falcon tubes
Isopropanol, 100% (Fisher, 10723124)
Ethanol, 100% (e.g. Fisher, 16606002)
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Tubos de ensayo Qubit™ (Invitrogen Q32856)
(Opcional) Seroalbúmina bovina (BSA) (50 mg/ml) (p. ej., Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Instrumental

Thermal cycler or heat block
Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
Mezclador vórtex
Microfuge
Wide-bore pipette tips
P1000 pipette and tips
Pipeta y puntas P200
Qubit fluorometer (or equivalent)
Ice bucket with ice
Timer

IMPORTANTE

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.

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

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

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:

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:

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. Computer requirements and software

Requisitos informáticos para el MinION Mk1B

Para secuenciar con el MinION Mk1B es necesario tener un ordenador o portátil de alto rendimiento, que pueda soportar la velocidad de adquisición de datos. Encontrará más información en el documento MinION Mk1B IT Requirements.

Requisitos informáticos para el MinION Mk1C

El MinION Mk1C contiene ordenador y pantalla integrados, lo que elimina la dependencia de cualquier accesorio para generar y analizar datos de nanoporos. Encontrará más información en el documento MinION Mk1C IT Requirements.

Software for nanopore sequencing

MinKNOW

The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.

For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.

EPI2ME (optional)

The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.

For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to the EPI2ME Platform protocol.

Verificar la celda de flujo

Antes de empezar el experimento de secuenciación, recomendamos verificar el número de poros disponibles, presentes en la celda de flujo. La comprobación deberá realizarse en los primeros tres meses desde su adquisición, si se trata de celdas de flujo MinION, GridION o PromethION, y en las primeras cuatro semanas tras la compra de celdas de flujo Flongle. Oxford Nanopore Technologies sustituirá cualquier celda de flujo con un número de poros inferior al indicado en la tabla siguiente, siempre y cuando el resultado se notifique dentro de los dos días siguientes a la comprobación y se hayan seguido las instrucciones de almacenamiento. Para verificar la celda de flujo, siga las instrucciones del documento Flow Cell Check.

Celda de flujo	Número mínimo de poros activos cubierto por la garantía
Flongle	50
MinION/GridION	800
PromethION	5000

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

Material

1.6 ml of whole blood

Consumibles

RBC Lysis Solution (QIAGEN, 158106)
Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
15 ml Falcon tubes
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

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.

CONSEJO

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

FIN DEL PROCESO

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

5. Preparation of tissue samples for gDNA extraction

Material

Cell Suspension Buffer (CSB): 0.35 M sucrose, 100 mM EDTA, 50 mM Tris.HCl pH 8
Frozen tissue sample

Consumibles

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

Instrumental

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

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

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

IMPORTANTE

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.

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.

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

CHECKPOINT

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

Assemble the pluriStrainer apparatus with a 100 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
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.
Disassemble the pluriStrainer® and retain the 100 µm strained homogenate in the 50 ml Falcon tube.

Assemble the pluriStrainer apparatus with a 50 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
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.
Disassemble the pluriStrainer® and retain the 50 µm strained homogenate in the 50 ml Falcon tube.

Assemble the pluriStrainer apparatus with a 30 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
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.
Disassemble the pluriStrainer® and retain the 30 µm strained homogenate in the 50 ml Falcon tube.

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

FIN DEL PROCESO

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

6. uHMW gDNA extraction

Material

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)

Consumibles

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)
Tubos de 1,5 ml Eppendorf DNA LoBind
2 ml Eppendorf DNA LoBind tubes

Instrumental

Heat block set at 56°C
Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
Mezclador Hula (mezclador giratorio suave)
Microfuge
Wide-bore pipette tips
P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips
Eppendorf 5424 centrifuge (or equivalent)

CONSEJO

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

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

Note: When using cell lines, we have found that this step can be omitted.

CONSEJO

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

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

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

CONSEJO

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

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

CONSEJO

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.

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

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

IMPORTANTE

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

IMPORTANTE

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

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

FIN DEL PROCESO

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

7. (Optional) gDNA quantification

Material

Monarch® DNA Capture Beads
Monarch® Bead Retainer
Monarch® Collection Tubes II

Consumibles

2 ml Eppendorf DNA LoBind tubes
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)

Instrumental

Mezclador vórtex
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.

CONSEJO

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.

CHECKPOINT

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

FIN DEL PROCESO

Take forward 750 µl DNA into the tagmentation step.

8. Tagmentation

Material

750 µl of extracted uHMW gDNA in EEB
Rapid Adapter (RA)
Fragmentation Mix (FRA)
FRA Dilution Buffer (FDB)

Consumibles

1.5 ml Eppendorf DNA LoBind tubes

Instrumental

Thermal cycler or heat block
Microfuge
Wide-bore pipette tips
P1000 pipette and tips
Pipeta y puntas P20
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.

For further information, please refer to the troubleshooting section.

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	✓	✓

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

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

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.

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

9. Clean-up

Material

Elution Buffer from the Oxford Nanopore kit (EB)
Precipitation Buffer (PTB)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Centrifuge
Microfuge
Mezclador Hula (mezclador giratorio suave)
P200 pipette and tips
P1000 pipette and tips
Wide-bore pipette tips

IMPORTANTE

The Precipitation Star (PS) found in the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) is currently not used for this method.

The method has been temporarily updated to omit the use of the Precipitation Star (PS) in the Clean-up step of this protocol. This is due to observations that using the Precipitation Star (PS) can lead to, in some cases, immediate pore loss after loading the ultra-long DNA library.

While we continue to investigate, this solution will prevent potential issues and has no negative impact on flow cell output, read length or pore occupancy.

For more information please visit this post in the Nanopore Community.

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.

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

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

FIN DEL PROCESO

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

CONSEJO

Recomendaciones de guardado de la biblioteca

Se recomienda guardar las bibliotecas en tubos Eppendorf DNA LoBind a 4 ⁰C, durante periodos de tiempo cortos o en caso de uso repetido, por ejemplo, para recargar celdas de flujo entre lavados. Para uso individual y para conservar a largo plazo por periodos de más de 3 meses, se recomienda guardar las bibliotecas a -80 ⁰C en tubos Eppendorf DNA LoBind.

10. Priming and loading the SpotON flow cell

Material

Flow Cell Flush (FCF) (enjuague de celda de flujo)
Flush Tether UL (FTU)
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind
Celda de flujo MinION/GridION
(Opcional) Seroalbúmina bovina (BSA) (50 mg/ml) (p. ej., Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Instrumental

Dispositivo MinION o GridION
Pantalla protectora para celdas de flujo MinION
Pipeta y puntas P1000
Pipeta y puntas P200
Pipeta y puntas P20

IMPORTANTE

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.

CONSEJO

Cebado y carga de la celda de flujo

Se recomienda a los nuevos usuarios que miren el vídeo Priming and loading your flow cell antes de realizar su primer experimento.

IMPORTANTE

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.

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.

MEDIDA OPCIONAL

Antes de cargar la biblioteca, verifique la celda de flujo para determinar el número de poros disponible.

Si se ha verificado con anterioridad la cantidad de poros presentes en la celda de flujo, este paso se puede omitir.

Dispone de más información en las instrucciones de comprobación de la celda de flujo, del protocolo de MinKNOW.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón. No retire más de 20-30 μl y asegúrese de que el tampón cubra la matriz de poros en todo momento. La introducción de burbujas de aire en la matriz puede dañar los poros de manera irreversible.

Ajustar una pipeta P1000 a 200 μl.
Introducir la punta de la pipeta en el puerto de cebado.
Girar la rueda hasta que el indicador de volumen marque 220-230 μl o hasta que se pueda ver una pequeña cantidad de tampón entrar en la punta de la pipeta.

Nota: Comprobar que haya un flujo continuo de tampón circulando desde el puerto de cebado a través de la matriz de poros.

IMPORTANTE

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.

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

IMPORTANTE

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.

Levantar suavemente la tapa del puerto de muestra SpotON.
Cargar 200 µl de mezcla de cebado en el puerto de cebado (no en el puerto de muestra SpotON), evitando introducir burbujas de aire.

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.

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.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, instale la pantalla protectora justo después de cargar la biblioteca.

Recomendamos poner la pantalla protectora en la celda de flujo y dejarla puesta mientras la biblioteca esté cargada, incluyendo los lavados y pasos de recarga. Retirar la pantalla cuando se haya extraído la biblioteca de la celda de flujo.

Colocar con cuidado el borde delantero de la pantalla protectora contra el clip. Nota: No hacer fuerza sobre ella.
Colocar la pantalla protectora con suavidad sobre la celda de flujo. La pieza debe asentarse alrededor de la tapa SpotON y debe cubrir por completo la sección superior de la celda de flujo.

ATENCIÓN

La pantalla protectora no está fijada a la celda de flujo. Una vez colocada, es necesario manipularla con cuidado.

FIN DEL PROCESO

Cerrar la tapa del dispositivo y configurar un experimento de secuenciación en MinKNOW.

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 modified method: Reloading ultra-long DNA library on a MinION/GridION flow cell.

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

Material

Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)
Flush Tether UL (FTU)
Flow Cell Flush (FCF) (enjuague de celda de flujo)
Sequencing Buffer UL (SBU)
Loading Solution UL (LSU)

Consumibles

1.5 ml Eppendorf DNA LoBind tubes
(Opcional) Seroalbúmina bovina (BSA) (50 mg/ml) (p. ej., Invitrogen™ UltraPure™ BSA 50 mg/ml, AM2616)

Instrumental

P1000 pipette and tips
Pipeta y puntas P200
Pipeta y puntas P20

IMPORTANTE

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.

CONSEJO

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.

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.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón. No retire más de 20-30 μl y asegúrese de que el tampón cubra la matriz de poros en todo momento. La introducción de burbujas de aire en la matriz puede dañar los poros de manera irreversible.

Ajustar una pipeta P1000 a 200 μl.
Introducir la punta de la pipeta en el puerto de cebado.
Girar la rueda hasta que el indicador de volumen marque 220-230 μl o hasta que se pueda ver una pequeña cantidad de tampón entrar en la punta de la pipeta.

Nota: Comprobar que haya un flujo continuo de tampón circulando desde el puerto de cebado a través de la matriz de poros.

IMPORTANTE

Para obtener un rendimiento de secuenciación óptimo y mejorar el rendimiento de las celdas de flujo MinION R10.4.1 (FLO-MIN114), recomendamos añadir seroalbúmina bovina (BSA), en una concentración total de 0,2 mg/ml, a la mezcla de cebado de la celda de flujo.

Nota: No se aconseja utilizar ningún otro tipo de albúmina (p. ej., seroalbúmina humana recombinante).

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

IMPORTANTE

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

IMPORTANTE

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.

Close the priming port and SpotON sample port cover, as indicated in the figure below.
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.

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.

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.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, instale la pantalla protectora justo después de cargar la biblioteca.

Colocar con cuidado el borde delantero de la pantalla protectora contra el clip. Nota: No hacer fuerza sobre ella.
Colocar la pantalla protectora con suavidad sobre la celda de flujo. La pieza debe asentarse alrededor de la tapa SpotON y debe cubrir por completo la sección superior de la celda de flujo.

ATENCIÓN

La pantalla protectora no está fijada a la celda de flujo. Una vez colocada, es necesario manipularla con cuidado.

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:

Pore scan:

12. Adquisición de datos e identificación de bases

Cómo empezar a secuenciar

Una vez cargada la celda de flujo, se puede iniciar el experimento en MinKNOW -el programa de secuenciación que gestiona el dispositivo, la adquisición de datos y la identificación de bases en tiempo real. Encontrará intrucciones de uso más detalladas en el protocolo de MinKNOW.

MinKNOW se puede configurar y utilizar para secuenciar de diferentes maneras:

En un ordenador conectado a un dispositivo de secuenciación, ya sea directamente o en remoto.
Directamente desde un dispositivo de secuenciación GridION, MinION Mk1C o PromethION 24/48.

Encontrará más información sobre el uso de MinKNOW en los manuales de usuario de los dispositivos:

Para empezar un experimento de secuenciación en MinKNOW:

1. Ir a la página de inicio y pulsar "Iniciar secuenciación".

2. Introducir los datos del experimento, como el nombre, la posición de la celda de flujo y el identificador de muestra.

3. En la pestaña Kit, seleccionar el kit de secuenciación usado durante la preparación de la biblioteca.

4. En las pestañas Run Options/Opciones de ejecución y Análisis, es posible configurar los parámetros de secuenciación adaptándolos al experimento o mantener la configuración predeterminada.

Nota: Si la identificación de bases estaba desactivada durante la configuración de un experimento, se puede activar en MinKNOW en la fase posejecución. Para más información, consulte el protocolo de MinKNOW.

5. En la pestaña Output/Datos de salida, es posible elegir entre configurar los parámetros de salida o mantener la configuración predeterminada.

6. En la pestaña Review/Revisar, revisar las opciones seleccionadas y pulsar Start/Empezar.

Análisis de datos tras la secuenciación

Una vez la secuenciación ha finalizado, la celda de flujo se puede reutilizar o devolver, como se indica en la sección Reutilización y devoluciones de celdas de flujo.

Tras la secuenciación y la identificación de bases, se puede proceder a analizar los datos. Si desea más información sobre las opciones de identificación de bases y de análisis posterior, consulte el documento Data Analysis.

En la sección Downstream analysis/Análisis posterior, le presentamos otras opciones para analizar los datos.

13. Reutilización y devoluciones de las celdas de flujo

Material

Flow Cell Wash Kit (EXP-WSH004) (kit de lavado de celda de flujo)

El protocolo Flow Cell Wash Kit está disponible en la comunidad Nanopore.

CONSEJO

Una vez terminado el experimento, recomendamos lavar la celda de flujo cuanto antes. Si no es posible, se puede dejar en el dispositivo y lavar al día siguiente.

Aquí puede encontrar las instrucciones para devolver celdas de flujo.

Nota: Antes de proceder a su devolución, las celdas de flujo deben lavarse con agua desionizada.

IMPORTANTE

Ante cualquier duda o pregunta acerca del experimento de secuenciación, consulte la guía de resolución de problemas, Troubleshooting Guide, que se encuentra en la versión en línea de este protocolo.

14. Análisis posterior

Análisis posterior a la identificación de bases

Existen varias opciones para completar el análisis de los datos de bases identificadas:

1. Plataforma EPI2ME

La plataforma EPI2ME es un servicio de análisis de datos, alojado en la red, desarrollado por Metrichor Ltd., filial de Oxford Nanopore Technologies. EPI2ME ofrece una serie de procesos de análisis, p. ej., de identificación metagenómica, identificación de especies a partir de un código (barcoding), alineación e identificación de variantes estructurales. El análisis no requiere equipo ni capacidad computacional extra y proporciona un informe fácil de interpretar con los resultados. Para obtener información sobre cómo realizar un proceso de análisis en EPI2ME, siga las instrucciones del protocolo, empezando por la sección "Starting data analysis".

2. EPI2ME Labs, tutoriales y procesos de trabajo

Para realizar un análisis de datos más exhaustivo, Oxford Nanopore Technologies ofrece una serie de tutoriales y procesos de trabajo de bioinformática, disponibles en EPI2ME Labs, que encontrará en la sección del mismo nombre de la comunidad Nanopore. La plataforma proporciona un espacio donde los proyectos que depositan en GitHub nuestros equipos de Investigación y Aplicaciones, se pueden exponer con textos descriptivos, código bioinformático funcional y datos de ejemplo.

3. Herramientas de análisis para la investigación

El departamento de Investigación de Oxford Nanopore Technologies ha creado una serie de herramientas de análisis que están disponibles en el repositorio Oxford Nanopore de GitHub. Las herramientas están diseñadas para usuarios avanzados y contienen instrucciones sobre cómo instalar y ejecutar el programa. Estas herramientas están públicamente disponibles y cuentan con un mantenimiento mínimo.

4. Herramientas de análisis desarrolladas por la comunidad

Si no se proporciona en ninguno de los recursos anteriores un método de análisis que responda a las necesidades de investigación requeridas, puede consultar la sección Bioinformatics del centro de recursos Resource Centre. Varios miembros de la comunidad Nanopore han desarrollado sus propias herramientas y segmentaciones para analizar datos de secuenciación por nanoporos. La mayoría de ellos está disponible en GitHub. Nótese que Oxford Nanopore Technologies no desarrolla ni mantiene esas herramientas y no garantiza que sean compatibles con la última configuración de química/software.

15. Issues during library preparation

A continuación hay una lista de los problemas más frecuentes, con algunas posibles causas y soluciones propuestas.

También disponemos de una página de preguntas frecuentes, FAQ, en la sección Support de la comunidad Nanopore.

Si ha probado las soluciones propuestas y continúa teniendo problemas, póngase en contacto con el departamento de asistencia técnica, bien por correo electrónico (support@nanoporetech.com) o a través del Live Chat de la comunidad Nanopore.

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.

Device:

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Explore products

Documentation

Nanopore Learning

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

Introduction to the protocol

Sample preparation

Library preparation

Secuenciación y análisis de datos

Troubleshooting

Document versions

Protocol

Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) VULK_9177_v114_revM_27Nov2022

Contents

Introduction to the protocol

Sample preparation

Library preparation

Secuenciación y análisis de datos

Troubleshooting

Overview

1. Overview of the protocol

IMPORTANTE

Este es un producto de acceso anticipado

Ultra-Long DNA Sequencing Kit features:

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

Flow cell loading and flushing

Best practice for handling uHMW gDNA

IMPORTANTE

Compatibility of this protocol

2. Equipment and consumables

Material

Consumibles

Instrumental

IMPORTANTE

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.

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

Flow Cell Wash Kit (EXP-WSH004) contents

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.

3. Computer requirements and software

Requisitos informáticos para el MinION Mk1B

Requisitos informáticos para el MinION Mk1C

Software for nanopore sequencing

MinKNOW

EPI2ME (optional)

Verificar la celda de flujo

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

Material

Consumibles

Instrumental

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

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.

CONSEJO

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.

FIN DEL PROCESO

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

5. Preparation of tissue samples for gDNA extraction

Material

Consumibles

Instrumental

Prepare the Cell Suspension Buffer (CSB) as follows:

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.

IMPORTANTE

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®.

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