PromethION: Protocol

Telomere-to-telomere sequencing (T2T) on PromethION (SQK-LSK114-XL and SQK-ULK114) V T2T_9179_v114_revL_31Jul2024

For Research Use Only

This is a Legacy protocol

Please use our new Telomere-to-telomere sequencing (T2T) on PromethION (SQK-APK114, SQK-LSK114, and SQK-ULK114) protocol for this application.

FOR RESEARCH USE ONLY

Descripción general

For Research Use Only

This is a Legacy protocol

Please use our new Telomere-to-telomere sequencing (T2T) on PromethION (SQK-APK114, SQK-LSK114, and SQK-ULK114) protocol for this application.

Document version: T2T_9179_v114_revL_31Jul2024

1. Overview of the protocol

IMPORTANTE

This is a Legacy protocol

Please use our new Telomere-to-telomere sequencing (T2T) on PromethION (SQK-APK114, SQK-LSK114, and SQK-ULK114) protocol for this application.

For more information on Nanopore-only Telomere-to-telomere (T2T) or to register your interest please follow this link.

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

Introduction to the protocol

This protocol describes an end-to-end workflow for telomere-to-telomere sequencing of the human genome using the Oxford Nanopore PromethION platform. The protocol includes three separate sequencing experiments, each requiring at least one PromethION Flow Cell, which are carried out over seven days. The library preparation, sequencing and flow cell washing and reloading steps are described day-by-day.

This protocol was developed in collaboration with the UCSC Nanopore Production Center, led by Dr. Karen Miga.

To generate as much data as possible, multiple flow cell washes are required to recover pores and reload fresh library to maximise data output. Depending on sample quality and DNA yield post-library preparation, the library preparation steps may need to be scaled up to meet the number of fresh libraries required for each flow cell wash and library reload. Excess DNA libraries can be stored in Eppendorf DNA LoBind tubes at 4°C until loading on the required day.

Three different datasets are generated to give both high accuracy duplex data and ultra-long reads, alongside the chromatin conformation capture data to achieve in-depth telomere-to-telomere sequencing of a sample.

Three experiments are set up across seven days:

Duplex experiment: This experiment generates high accuracy duplex data using the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL). Library preparation takes ~60 minutes hands-on time, followed by three washes across a 100-hour sequencing run.
Ultra-long DNA experiment: This experiment generates ultra-long reads using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114). DNA extraction and library preparation takes ~3.5 hours with an overnight elution, followed by two washes across a 72-hour sequencing run. This experiment generates a very viscous library of very long DNA fragments which requires careful handling to maintain the long fragments.
Pore-C experiment: This experiment produces chromatin conformation capture data using the Pore-C protocol and the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL). The Pore-C DNA extraction takes ~3 hours hands-on time over three days with two overnight steps. The library preparation takes ~60 minutes hands-on time, followed by three washes across a 72-hour sequencing run. This experiment has been developed by Oxford Nanopore Technologies and the following published literature: Lieberman-Aiden et al., 2009; Comet et al., 2011; Belton et al., 2012; Gavrilov, Golov and Razin, 2013; Nagano et al., 2015; Belaghzal, Dekker and Gibcus, 2017; Ulahannan et al., 2019. This experiment intends to manipulate cell suspensions extracted from whole blood to capture three-dimensional interactions of DNA within chromatin. This workflow has been written using NlaIII restriction enzyme and the heat denaturation method. For further information on protocol considerations, please see the Restriction Enzyme Pore-C info sheet.

Steps in the sequencing workflow

Prepare for your experiment You will need to:

Prepare your samples for all experiments.
Ensure you have your sequencing kits, the correct equipment and third-party reagents.
Download the software for acquiring and analysing your data.
Check your flow cells to ensure they have enough pores for a good sequencing run.

Library preparation

Duplex experiment: You will need to:

Repair the DNA, and prepare the DNA ends for adapter attachment
Attach sequencing adapters to the DNA ends
Prime the flow cell, and load your DNA library into the flow cell
Wash and reload the flow cell approximately every 20-24 hours

__Ultra-long DNA experiment:__ You will need to:

Extract your uHMW gDNA
Tagment your DNA using a diluted fragmentation mix
Attach the sequencing adapters to the DNA ends
Clean-up the sample by precipitating your DNA and elute overnight
Prime the flow cell and load your DNA library into the flow cell
Wash and reload the flow cell approximately every 20-24 hours

__Pore-C experiment:__ You will need to:

Crosslink the three dimensional DNA interactions within the nucleus
Permeabilise the cells to expose the crosslinked structures and denature the chromatin
Cleave the genome into clusters of crosslinked DNA fragments at recognition sites with a restriction enzyme
Ligate the cohesive ends of proximal crosslinked monomers into chimeric Pore-C polymers held in proximity
Degrade the protein structures to release the chimeric Pore-C polymers into solution
Purify the Pore-C extract before DNA repair and end-prep for adapter attachment
Attach sequencing adapters to the DNA ends
Prime the flow cell, and load your DNA library into the flow cell
Wash and reload the flow cell approximately every 18 hours

Sequencing and analysis

You will need to:

For each experiment, start a sequencing run using the MinKNOW software which will collect raw data from the device and convert it into basecalled reads.
For each experiment, we recommend basecalling in real-time during sequencing before rebasecalling and aligning to a reference genome post-sequencing. Further details for each experiment set up are outlined in the "Sequencing and data analysis" section.

IMPORTANTE

Compatibility of this protocol

This protocol should only be used in combination with:

Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
Flow Cell Wash Kit XL (EXP-WSH004-XL)
Flow Cell Priming Kit V14 (EXP-FLP004)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
R10.4.1 PromethION Flow Cells (FLO-PRO114M)

2. Equipment and consumables

Material

>15 ml of whole blood
Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
Flow Cell Wash Kit XL (EXP-WSH004-XL)

Consumibles

PromethION Flow Cell (FLO-PRO114M)
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
Monarch® HMW DNA Extraction Kit for Tissue (NEB, T3060)
Puregene Blood Kit (QIAGEN, 158023)
T4 DNA Ligase 400,000 U/ml (NEB, M0202S/L)
NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB E7180S or E7180L) (módulo de acompañamiento NEBNext de secuenciación por ligación para Oxford Nanopore Technologies®) Como alternativa, se pueden utilizar los siguientes productos de NEBNext®:
NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
NEBNext FFPE DNA Repair Mix (NEB M6630)
RBC Lysis Solution (QIAGEN, 158106)
Agencourt AMPure XP beads (Beckman Coulter, A63881)
5 M NaCl (Sigma, 71386)
PEG 8000, 50% w/v (Rigaku Reagents, 25322-68-3)
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
Percoll, 1.135 g/ml (Cytiva, 17-0891-01)
(Optional) dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 20-139)
ECOSURF EH-9 (Dow, 64366-70-7)
Fetal bovine serum (FBS) (Gibco™, A3840401)
Glycine (Sigma, 56-40-6)
Formaldehyde at 36.5% v/v (Sigma, 33220)
NlaIII restriction enzyme with CutSmart Buffer (NEB, R0125L)
IGEPAL CA-630 (Sigma, I8896)
Protease Inhibitor Cocktail (Sigma, P8340)
Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
Tween-20 (Thermo Scientific, J20605.AP)
1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
Proteinase K at 20 μg/μl (NEB, P8107S)
10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
Recombinant Albumin at 20 μg/μl (NEB, B9200S)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Ethanol, 100% (e.g. Fisher, 16606002)
Isopropanol, 100% (Fisher, 10723124)
Chilled phenol:chloroform:isoamyl alcohol in a 25:24:1 ratio, saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA (Sigma, P3803-400ML)
3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
50 ml centrifuge tubes
15 ml Falcon tubes
2 ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubes
Tubos de ensayo Qubit™ (Invitrogen Q32856)
Ziplock bags
0.2 µm filter

Instrumental

Dispositivo PromethION 24/48
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P100
Pipeta y puntas P10
Pipeta y puntas P20
Wide-bore pipette tips
Pasteur pipettes
Thermal cycler or heat block
Mezclador Hula (mezclador giratorio suave)
Gradilla magnética
Vortex mixer
Temperature-controlled centrifuge
Microfuge
Cubeta con hielo
Thermomixer
Fluorímetro Qubit (o equivalente para el control de calidad)
Class I hood with active charcoal filter
-80°C freezer storage

Equipo opcional

Liquid nitrogen and canister

Approximately 15 ml of whole blood is required as input for the sample preparation steps.

Aliquots of whole blood are prepared for each experiment to isolate the peripheral blood mononuclear cells (PBMCs) for the Pore-C and Ultra-long DNA experiments and to extract gDNA for the Duplex experiment.

The whole blood can be collected in an anticoagulant such as K2-EDTA but we do not recommend mixing with other additives as they may interfere with the Pore-C DNA extraction or the DNA sequencing run.

We recommend different sample preparations due to different input requirements for each experiment. However, users may use other methods they feel are most appropriate but to ensure the individual input requirements are followed. Depending on how DNA is extracted from a sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page.

Input requirements:

Pore-C experiment: 10 million PBMCs
Duplex experiment: 1 μg or 100-200 fmol extracted DNA
Ultra-long DNA experiment: 6 million PBMCs

We recommend preparing your samples and the custom SPRI bead suspension a day ahead of the experiments to ensure maximum use of time each day.

Reactivos de otros fabricantes

Oxford Nanopore Technologies ha probado y recomienda el uso de todos los reactivos de otros fabricantes citados en este protocolo. No se han evaluado otras alternativas.

Recomendamos preparar estos reactivos siguiendo las instrucciones del fabricante.

NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

We recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L). This contains all the NEB reagents needed for use with the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) during day 1 of the Duplex experiment and day 4 of the Pore-C experiment.

Please note, the Pore-C experiment also requires additional use of the T4 DNA Ligase kit for the proximity ligase reaction during day 2 of the protocol.

Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents

Name	Acronym	Vial colour	Number of vials	Fill volume per vial (µl)
DNA Control Strand	DCS	Yellow	1	100
Ligation Adapter	LA	Green	1	320
Ligation Buffer	LNB	White	1	1,500
Elution Buffer	EB	White cap, black strip label	1	10,000
Long Fragment Buffer	LFB	White cap, orange strip label	2	20,000
Short Fragment Buffer	SFB	White cap, blue strip label	2	20,000
Library Beads	LIB	Pink	2	1,800
Library Solution	LIS	White cap, pink label	2	1,800
Sequencing Buffer	SB	Red	3	1,700
Flow Cell Flush	FCF	Clear	4	15,500
Flow Cell Tether	FCT	Purple	1	1,600

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

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

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:

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 XL (EXP-WSH004-XL) contents

Name	Acronym	Fill volume per vial (µl)	Cap colour	No. of vials	No. of uses
Wash Mix	WMX	150	Brown	1	48
Wash Diluent	DIL	20,000	White cap, brown stripe	1	48
Storage Buffer	S	25,000	White cap, yellow stripe	1	48

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.

3. Computer requirements and software

PromethION 24/48 IT requirements

The PromethION device contains all the hardware required to control up to 24 (for the P24 model) or 48 (for the P48 model) sequencing experiments and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the PromethION IT Requirements document.

PromethION 2 Solo IT requirements

The PromethION 2 (P2) Solo is a device which directly connects into a GridION Mk1 or a stand-alone computer that meets the miminum specifications for real-time data streaming and analysis. Up to two PromethION flow cells can be can be run and each is independently addressable, meaning experiments can be run concurrently or individually. For information on the computer IT requirements, please see the PromethION 2 Solo IT requirements document.

Check your PromethION flow cell

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for PromethION flow cells. Oxford Nanopore Technologies will replace any flow cell with fewer than 5000 pores when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Programas informáticos para secuenciar por nanoporos

MinKNOW

El programa MinKNOW controla el dispositivo de secuenciación por nanoporos, genera los datos de secuenciación e identifica las bases en tiempo real. Se utiliza para secuenciar y demultiplexar las muestras, cuando estas contienen códigos.

Para ponerlo en funcionamiento, consulte el protocolo MinKNOW en la sección correspondiente.

EPI2ME (opcional)

La plataforma en la nube EPI2ME realiza análisis adicionales de las bases identificadas, por ejemplo la alineación con el genoma lambda, identificación de especies mediante código de barras (barcoding) o clasificación taxonómica. EPI2ME es necesario solo si tras la identificación de bases se necesitan análisis adicionales.

Para crear una cuenta en EPI2ME e instalar el agente de escritorio, consulte el protocolo de la plataforma.

4. Custom SPRI bead preparation for the Pore-C experiment

Consumibles

Agencourt AMPure XP beads (Beckman Coulter, A63881)
1 M Tris-HCl, pH 7.5
0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
5 M NaCl (Sigma, 71386)
PEG 8000, 50% w/v (Rigaku Reagents, 25322-68-3)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Etanol al 80 % recién preparado con agua sin nucleasas
2 ml Eppendorf DNA LoBind tubes

Instrumental

Gradilla magnética
Mezclador Hula (mezclador giratorio suave)
Thermal cycler or heat block
P1000 pipette and tips
Pipeta y puntas P200
P100 pipette and tips
Pipeta y puntas P10
Wide-bore pipette tips

Custom SPRI bead suspension preparation for Pore-C extraction

Before starting the Pore-C experiment, a custom SPRI bead suspension needs to be prepared. This will be used to deplete non-chimeric monomers and to maximise the frequency of chimeric Pore-C polymers, improving purity ratios and read lengths at the end of Day 3: Pore-C experiment.

We also recommend using this custom SPRI bead suspension to size-select the extracted gDNA input in the Whole blood sample gDNA extraction for the Duplex experiment to enrich for fragments above 1.5-2 kb.

Store the beads at 4°C and bring to room temperature before use.

Reagent	Final	Volume
Tris-HCl, 1 M	10 mM	20 μl
EDTA, pH 8, 0.5 M	1 mM	4 μl
NaCl, 5 M	1.6 M	640 μl
PEG 8000, 50% (w/v)	11% (w/v)	440 μl
Nuclease-free water	-	888 μl
Total	-	1992 μl

Note: We recommend using wide-bore 1 ml pipette tips to accurately pipette 440 μl of 50% PEG 8000.

FIN DEL PROCESO

Store the beads at 4°C. Before use, bring the suspension to room temperature.

5. Whole blood sample preparation for the Pore-C experiment

Material

5–10 ml whole blood

Consumibles

10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
Percoll, 1.135 g/ml (Cytiva, 17-0891-01)
(Optional) dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 20-139)
Fetal bovine serum (FBS) (Gibco™, A3840401)
(Optional) chilled fetal bovine serum (FBS) (Gibco™, A3840401)
50 ml centrifuge tubes
2 ml Eppendorf DNA LoBind tubes

Instrumental

Pasteur pipettes
Temperature-controlled centrifuge with rotor for 2 ml and 50 ml tubes
Cubeta con hielo
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P100
Pipeta y puntas P20
Pipeta y puntas P10

Equipo opcional

Liquid nitrogen and canister
-80°C freezer storage

PBMC sample preparation for Pore-C DNA extraction

Before starting the Pore-C DNA extraction, the peripheral blood mononuclear cells (PBMCs) must be isolated from whole blood whilst maintaining cell viability. Approximately 5–10 ml of whole blood should yield sufficient PBMCs for the Pore-C DNA extraction. If necessary, combine multiple aliquots of whole blood to achieve a final 5–10 ml pooled sample. Using the below method, approximately 10 million PBMCs are prepared in aliquots of 1X PBMCs supplemented with 2% FBS. Approximately 10 million PBMCs are taken forwards into the day 1 of the Pore-C experiment.

Users may isolate PBMCs by any means they feel are most appropriate for the whole blood sample to be used, provided that:

PBMCs are isolated as soon as possible from fresh blood and no later than 24 hours.
PBMC are isolated using a method optimised for cell viability.
The whole blood is not mixed with any additives, except for anticoagulants (e.g. K2-EDTA), which are acceptable and will not interfere with the Pore-C DNA extraction.

If users have a minimum of approximately 10 million PBMCs which have been isolated previously which satisfy these requirements, they may start sample extraction directly from day 1 of the Pore-C experiment.

500 ml of 1X PBS supplemented with 2% FBS final concentration and store at room temperature.

Reagent	Volume
10X PBS	50 ml
Fetal bovine serum (FBS)	10 ml
Nuclease-free water	440 ml
Total	500 ml

100 ml of 1X PBS supplemented with 60% Percoll final concentration and store at room temperature.

Reagent	Volume
10X PBS	10 ml
Percoll	60 ml
Nuclease-free water	30 ml
Total	100 ml

(Optional for storage) 2 ml of FBS supplemented with 20% DMSO final and store at 4°C.

Reagent	Volume
FBS	1,600 µl
DMSO	400 µl
Total	2,000 µl

MEDIDA OPCIONAL

The cells can be stored if Pore-C sample extraction cannot be started immediately.

Centrifuge at 350 x g at 4°C for 2 minutes with the brake on.
Aspirate and discard the supernatant, then resuspend the white blood cells pellet in 1 ml of chilled FBS.
Once resuspended, slowly mix in 1 ml of chilled FBS supplemented with 20% DMSO, drop by drop.
Note: As DMSO is mixed with water, energy is released as heat. Adding DMSO to the white blood cells suspension drop by drop prevents heat shock to the cells.
Snap freeze aliquots of white blood cells in liquid nitrogen then store at –80°C.

FIN DEL PROCESO

Take forward approximately 10 million white blood cells into the Pore-C experiment. Store the cells at 4°C until the experiment can begin.

6. Whole blood sample gDNA extraction for the Duplex experiment

Material

3 ml of whole blood
Custom SPRI bead suspension

Consumibles

Puregene Blood Kit (QIAGEN, 158023)
Etanol al 80 % recién preparado con agua sin nucleasas
Isopropanol, 100% (Fisher, 10723124)
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
Tubos de 1,5 ml Eppendorf DNA LoBind
15 ml Falcon tubes
Tubos de ensayo Qubit™ (Invitrogen Q32856)
Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)

Instrumental

Centrifuge and rotor suitable for 15 ml Falcon tubes
Heating block
Mezclador vórtex
Fluorímetro Qubit (o equivalente para el control de calidad)

Whole blood gDNA extraction for the Duplex experiment

An input of 1 µg of gDNA must be prepared for the Duplex experiment. You may extract the gDNA by any means is most appropriate. This section outlines how to extract gDNA from whole blood using the QIAGEN Puregene Blood Kit.

Dispense 9 ml of RBC Lysis Solution into a 15 ml Falcon tube.
Add 3 ml of whole blood and mix by inverting 10 times.
Incubate for 5 minutes at room temperature. Invert at least once during the incubation.
Centrifuge for 2 minutes at 2000 x g to pellet the white blood cells.
Carefully discard the supernatant by pipetting or pouring, leaving approximately 200 µl of the residual liquid and the white blood cell pellet.
Vortex the tube vigorously to resuspend the pellet in the residual liquid. Vortexing greatly facilitates cell lysis in the next step.
Add 3 ml of Cell Lysis Solution and pipette mix to lyse the cells or vortex for 10 seconds.

Add 15 µl of RNase A Solution and mix by inverting 25 times. Incubate for 15 minutes at 37°C. Then incubate for 3 minutes on ice to quickly cool the sample.
Add 1 ml of Protein Precipitation Solution and vortex vigorously for 20 seconds at high speed.
Centrifuge for 5 minutes at 2000 x g. The precipitated proteins should form a tight brown pellet. If the protein pellet is not tight, incubate on ice for 5 minutes and repeat the centrifugation.
Pipette 3 ml of isopropanol into a clean 15 ml Falcon tube and add the supernatant from the previous step by pouring carefully. Be sure that the protein pellet is not dislodged during pouring.
Mix by inverting 50 times until the DNA is visible as threads or a clump.
Centrifuge for 3 minutes at 2000 x g. The DNA may be visible as a small white pellet.
Carefully discard the supernatant and drain the tube by inverting on a clean piece of absorbent paper, taking care that the pellet remains in the tube.
Add 3 ml of 80% ethanol and invert several times to wash the DNA pellet.
Centrifuge for 1 minute at 2000 x g.
Carefully discard the supernatant. Drain the tube on a clean piece of absorbent paper, taking care that the pellet remains in the tube. Dry the pellet for 5-10 minutes. The pellet might be loose and easily dislodged. Avoid over-drying the DNA pellet as the DNA will be difficult to dissolve.

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

CHECKPOINT

Quantify 1 µl of size-selected DNA using a Qubit fluorometer.

You can expect a 50-55% loss of DNA depending on a fragment length distribution of input material: the greater the proportion of short fragments (<1.5-2 kb), the greater the sample loss.

FIN DEL PROCESO

Take forward 1 µg of extracted DNA into the Duplex experiment. Store at 4°C until the experiment can begin.

7. Whole blood sample preparation for the Ultra-long DNA experiment

Material

1.6 ml of whole blood

Consumibles

RBC Lysis Solution (QIAGEN, 158106)
10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
15 ml Falcon tubes
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Temperature-controlled microfuge
P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips

PBMC sample preparation for the Ultra-long DNA experiment

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

Users may isolate PBMCs 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 day 1 of the Ultra-long DNA experiment.

Reagent	Volume
10X PBS	1 ml
Nuclease-free water	9 ml
Total	10 ml

CONSEJO

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

FIN DEL PROCESO

Take forward 6 million PBMCs forward into the Ultra-Long DNA experiment. Store the pellet at 4°C until the experiment can begin.

8. Day 1: Pore-C experiment

Material

10 million white blood cells isolated from whole blood

Consumibles

Nuclease-free water (e.g. ThermoFisher, AM9937)
NlaIII restriction enzyme with CutSmart Buffer (NEB, R0125L)
ECOSURF EH-9 (Dow, 64366-70-7)
Glycine (Sigma, 56-40-6)
Formaldehyde at 36.5% v/v (Sigma, 33220)
IGEPAL CA-630 (Sigma, I8896)
Protease Inhibitor Cocktail (Sigma, P8340)
Chilled 10X phosphate-buffered saline (PBS) (Thermo Fisher, 70011044)
Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
5 M NaCl (Sigma, 71386)
1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
Ziplock bags
0.2 µm filter
50 ml centrifuge tubes
2 ml Eppendorf DNA LoBind tubes
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Class I hood with active charcoal filter
Temperature-controlled centrifuge
Thermal cycler or heat block
Thermomixer
Vortex mixer
Mezclador Hula (mezclador giratorio suave)
Cubeta con hielo
P1000 pipette and tips
Pipeta y puntas P100
P200 pipette and tips
Pipeta y puntas P20
Wide-bore pipette tips

Day 1: Pore-C experiment overview

During day 1, the PBMCs are prepared for stabilising of the three-dimensional interactions of DNA in the nucleus by chemically cross-linking DNA and protein. The nuclei are then permeabilised to expose the crosslinked cytoskeleton cage and nuclear structures before the chromatin is denatured. The DNA is now accessible to the chosen restriction enzyme† which passively diffuses through the crosslinked cytoskeleton cage and nuclear structures to digest the genome at compatible recognition sites. The sample is incubated overnight which creates clusters of DNA fragments held in proximity by crosslinks between DNA and the cytoskeleton, preserving the original interactions which were crosslinked.

†This protocol has been written using NlaIII and the heat denaturation method as our investigations have found this 4-cutter is particularly suitable for Pore-C across many different species, yielding Pore-C extracts with high contact densities. For more information, please see the "Protocol considerations" section of our Restriction enzyme Pore-C info sheet.

Thaw both reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the NlaIII restriction enzyme.
Spin down tubes before opening for the first time each day.

Reagent	Volume
10% SDS	100 µl
Nuclease-free water	900 µl
Total	1,000 µl

Weigh out 1 g of ECOSURF™ EH-9.
Transfer to a fresh 15 ml Falcon tube.
Add 9 ml of nuclease-free water.
Gently pipette mix with a wide-bore pipette tip until the solution is homogenous.

ATENCIÓN

1% formaldehyde solution is a biological hazard. Formaldehyde crosslinks DNA and is a mutagen. It must be handled with caution, and vessels containing the solution should only be uncapped in a class I hood.

Transfer 10 ml of 1X PBS into a 50 ml Falcon tube. Note: Using a 15 ml Falcon tube is not recommended.
Inside a class I hood, with double gloves, add 291 μl of 36.5% formaldehyde to the 10 ml 1X PBS aliquot to a final concentration of 1% formaldehyde in ~10.3 ml.
Mix by gentle inversion, and open the tube to allow gases to escape, then close the tube.
Check that no formaldehyde residue has remained on the gloves, Falcon tube, or pipette.
Remove the outer gloves and discard them in a biohazard bag in the hood.
Remove the 1% formaldehyde 1X PBS solution from the hood.
Store the tube with formaldehyde inside a zip lock bag at 4°C prior to use.

Take approximately 10 million white blood cells and briefly homogenise the suspension by gently pipetting with a wide-bore pipette tip.
Transfer the cell suspension to a 50 ml centrifuge tube.
Rinse the original tube with a further 1 ml of chilled 1X PBS into the 50 ml centrifuge tube.
Bring the volume of the resuspended white blood cells to 10 ml in chilled 1X PBS.
Proceed with the Pore-C experiment using approximately 10 million white blood cells as input.

IMPORTANTE

We do not recommend extending incubation times as it may have a detrimental impact on the efficiency of de-crosslinking the DNA later in the protocol.

Note: 2 ml Eppendorf tubes are required for a compact sample pellet. Do not use 1.5 ml tubes.

CONSEJO

The rest of the protocol can be continued outside of the class I hood.

IMPORTANTE

Process each crosslinked sample pellet separately. Do not pool multiple pellets into a single reaction.

The remainder of the protocol is written for one pellet.

MEDIDA OPCIONAL

We advise continuing with a freshly crosslinked sample pellet. However, if you intend to store samples for later use, you can snap-freeze the aliquots in liquid nitrogen. Store frozen sample pellets at –80°C and use within one year.

IMPORTANTE

Do not proceed any further unless it is possible to complete the remainder of this section consecutively without interruption. It is not advisable to incubate any step longer than stated in this protocol. Doing so may be detrimental to Pore-C data quality and sequencing performance.

Reagent	Volume
Nuclease-free water	510 µl
10X CutSmart Buffer	90 µl
Total	600 µl

Reagent	Final	Volume
Tris-HCl, pH 8.0, 1 M	10 mM	5 µl
NaCl, 5 M	10 mM	1 µl
IGEPAL CA-630, 10%	0.2%	10 µl
Nuclease-free water	-	484 µl
Total	-	500 µl

IMPORTANTE

Following centrifugation, the pellet will be delicate. Carefully aspirate and discard as much of the supernatant as possible without disturbing the pellet

The SDS may precipitate at this point; this will not impact the experiment so proceed to the next step.

Note: This incubation can be performed without mixing.

The SDS may precipitate at this point. This will not impact the experiment so proceed to the next step.

Reagent	Final	Volume
Permeabilised cells	-	371 µl
NEB NlaIII, 10 U/µl	1 U/µl	45 µl
Nuclease-free water	-	34 µl
Total	-	450 µl

Note: This incubation can be performed without mixing.

FIN DEL PROCESO

During the Pore-C incubation, start the Duplex experiment.

9. Day 1: Duplex experiment

Material

1 μg or 100-200 fmol DNA
Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)

Consumibles

NEBNext FFPE DNA Repair Mix (NEB M6630)
NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
Agua sin nucleasas (p. ej., ThermoFisher AM9937)
Etanol al 80 % recién preparado con agua sin nucleasas
Tubos de ensayo Qubit™ (Invitrogen Q32856)
Tubos de 1,5 ml Eppendorf DNA LoBind
Tubos de PCR de pared fina (0,2 ml)

Instrumental

Pipeta y puntas P1000
Pipeta y puntas P100
Pipeta y puntas P20
Pipeta y puntas P10
Microcentrífuga
Mezclador Hula (mezclador giratorio suave)
Gradilla magnética
Cubeta con hielo
Mezclador vórtex
Termociclador
Fluorímetro Qubit (o equivalente para el control de calidad)

Day 1: Duplex experiment overview

In this step, the library for the Duplex experiment is prepared for sequencing, as follows: the extracted DNA is repaired and the ends prepared for adapter attachment using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-tailing Module reagents. The sequencing adapters are attached to the DNA fragment ends before a clean-up step in preparation for sequencing on the PromethION Flow Cell.

CONSEJO

Se recomienda usar DNA Control Sample (DCS) en la preparación de la biblioteca para localizar y solucionar problemas. De todos modos, se puede omitir este paso y compensar el 1 µl extra con la muestra de ADN.

Para obtener un rendimiento óptimo, NEB recomienda lo siguiente:

Descongelar todos los reactivos en hielo.
Golpear suavemente los tubos de reactivos con el índice o invertirlos, para asegurarse de que estén bien mezclados.
Nota: No mezclar en vórtex las mezclas FFPE DNA Repair Mix, ni Ultra II End Prep Enzyme Mix.
Centrifugar los tubos antes de abrirlos.
Los tampones Ultra II End Prep Buffer y FFPE DNA Repair Buffer pueden tener un poco de precipitado. Dejar que la mezcla alcance la temperatura ambiente y mezclar pipeteando varias veces para romper el precipitado; para solubilizarlo, agitar el tubo en vórtex durante 30 s.
Nota: Es importante mezclar bien los tampones mediante vórtex.
El tampón FFPE DNA Repair Buffer puede tener un matiz amarillo; no importa si está así; se puede utilizar.

Transfer 1 μg (or 100-200 fmol) input DNA into a 1.5 ml Eppendorf DNA LoBind tube.
Adjust the volume to 47 μl with nuclease-free water.
Mix thoroughly by pipetting up and down, or by flicking the tube.
Spin down briefly in a microfuge

Entre cada adición, mezclar con la pipeta de 10 a 20 veces.

Reactivo	Volumen
ADN del paso anterior	47 µl
(opcional) DNA Control Sample (DCS)	1 µl
NEBNext FFPE DNA Repair Buffer	3,5 µl
NEBNext FFPE DNA Repair Mix	2 µl
Ultra II End-prep Reaction Buffer	3,5 µl
Ultra II End-prep Enzyme Mix	3 µl
Total	60 µl

CHECKPOINT

Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit.

MEDIDA OPCIONAL

If a pause is required, the sample can be stored overnight at 4°C.

IMPORTANTE

Aunque la ligasa recomendada de otros fabricantes se suministra con su propio tampón, la eficiencia del adaptador, Ligation Adapter (LA), es mayor cuando se usa el tampón Ligation Buffer (LNB) suministrado en el kit Ligation Sequencing Kit.

Entre cada adición, mezclar con la pipeta de 10 a 20 veces.

Reactivo	Volumen
Muestra de ADN del paso anterior	60 µl
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Ligation Adapter (LA)	5 µl
Total	100 µl

Dispose of the pelleted beads

CHECKPOINT

Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit.

IMPORTANTE

We recommend loading 10-20 fmol of this final prepared library onto the R10.4.1 flow cell.

Loading more than 20 fmol of DNA can reduce the rate of duplex read capture. Dilute the library in Elution Buffer if required.

FIN DEL PROCESO

Take the 32 µl of the library forwards for loading onto the flow cell. Store on ice until ready to load.

10. Day 1: Priming and loading Duplex library on the PromethION Flow Cell

Material

Sequencing Buffer (SB)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Library Solution (LIS)
Flow Cell Tether (FCT) (anclaje de celda de flujo)
Flow Cell Flush (FCF)

Consumibles

PromethION Flow Cell (FLO-PRO114M)
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Dispositivo PromethION 24/48
PromethION Flow Cell Light Shield
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P20

Day 1: Duplex experiment flow cell loading

Once the library has been prepared, the PromethION Flow Cell can be primed before the library is combined with the sequencing reagents and loaded into the flow cell.

We recommend monitoring your sequencing run and to reload your flow cell when recommended to in the "Washing and reloading Duplex library on PromethION Flow Cells". The Duplex experiment can be reloaded three times, across days 2, 3 and 4.

Uso de Library Solution (LIS)

En la mayoría de experimentos de secuenciación, recomendamos usar Library Beads (LIB) para cargar la biblioteca en la celda de flujo. Nótese, si previamente se ha usado agua para cargar la biblioteca, se deberá usar Library Solution (LIS) en su lugar. Nota: Algunos clientes han notado que las bibliotecas viscosas pueden cargarse con mayor facilidad cuando no se usan Library Beads (LIB).

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Una vez sacadas de la nevera, esperar 20 minutos antes de insertar las celdas de flujo en el dispositivo y así darles tiempo a que estén a temperatura ambiente. En entornos húmedos se puede formar condensación. Inspeccione las clavijas doradas del conector, situadas en la parte superior e inferior de la celda de flujo, en busca de condensación y si la hubiera, límpiela con una toallita sin pelusa. Procure que la almohadilla térmica (color gris oscuro) esté enganchada en la parte posterior.

Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

IMPORTANTE

Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Recomendamos colocar 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.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

FIN DEL PROCESO

Close the PromethION lid when ready to start a sequencing run on MinKNOW.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

11. Day 2: Pore-C experiment

Consumibles

T4 DNA Ligase 400,000 U/ml (NEB, M0202S/L)
Recombinant Albumin at 20 μg/μl (NEB, B9200S)
Tween-20 (Thermo Scientific, J20605.AP)
Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
Proteinase K at 20 μg/μl (NEB, P8107S)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Tubos de 1,5 ml Eppendorf DNA LoBind
2 ml Eppendorf DNA LoBind tubes

Instrumental

Thermomixer
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P100
P20 pipette and tips

Day 2: Pore-C experiment overview

For Day 2, after the overnight incubation, the restriction enzymes are heat inactivated to prevent re-digesting ligated products. DNA ligase is added to the clusters of crosslinked DNA and passively diffuses through the crosslinked cytoskeleton cage to ligate the cohesive ends of proximal monomers into chimeric Pore-C polymers. After ligation, the ligated products can be released from the crosslinked cytoskeleton cages by an overnight proteinase K digestion. This releases the chimeric Pore-C polymers into solution as dsDNA.

Thaw the reagents on ice.
Flick and/or invert the reagent tube(s) to ensure they are well mixed.
Note: Do not vortex the T4 DNA Ligase enzyme.
Spin down tubes before opening for the first time each day.

Weigh out 1.095 g of Tween-20 and transfer to a fresh 5 ml centrifuge tube.
Add 4 ml of nuclease-free water.
Gently invert the tube until the solution is homogenous.

Reagent	Final	Volume
Digestion reaction (from Day 1)	-	450 µl
Nuclease-free water	-	395 µl
T4 DNA Ligase Reaction Buffer, 10X	1X	100 µl
Recombinant albumin, 20 µg/µl	0.1 µg/µl	5 µl
T4 DNA Ligase, 400 U/µl	20 U/µl	50 µl
Total	-	1000 µl

Note: This incubation can be performed without mixing.

IMPORTANTE

Do not extend incubation as prolonged ligation may increase trans-chromosomal contacts in the Pore-C data.

CONSEJO

During this 6 hour incubation, the Ultra-long DNA experiment may be started.

Reagent	Final	Volume
Ligation reaction (from the Proximity Ligation)	-	1000 μl
Nuclease-free water	-	300 μl
Tween-20, 20%	5%	500 μl
SDS, 10%	0.5%	100 μl
Proteinase K, 20 μg/μl	1 μg/μl	100 μl
Total	-	2000 μl

Note: This incubation can be performed without mixing.

IMPORTANTE

Incubation at 56°C compromises enzyme activity over a prolonged incubation. It is not advisable to incubate at higher temperatures as enzyme activity will reduce over time.

FIN DEL PROCESO

During the Pore-C incubation, start the Ultra-long DNA experiment.

12. Day 2: Ultra-long DNA experiment

Material

6 million PBMCs isolated from whole blood
Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)

Consumibles

Monarch® HMW DNA Extraction Kit for Tissue (NEB, T3060)
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
Isopropanol, 100% (Fisher, 10723124)
Ethanol, 100% (e.g. Fisher, 16606002)
5 ml Eppendorf DNA LoBind tubes
2 ml Eppendorf DNA LoBind tubes
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Termociclador
Temperature-controlled centrifuge
Microfuge
Mezclador Hula (mezclador giratorio suave)
Vortex mixer
Qubit fluorometer (or equivalent)
Pipeta y puntas P1000
P200 pipette and tips
Pipeta y puntas P20
Wide-bore pipette tips
Cubeta con hielo

Day 2: Ultra-long DNA experiment overview

During the first day of the Ultra-long DNA experiment, the ultra-high molecular weight (uHMW) gDNA is extracted using the NEB Monarch HMW DNA Extraction Kit for Tissue. An optional uHMW gDNA quantification step has also been included. However, this step can be omitted and 750 µl of DNA in Extraction EB (EEB) can be taken straight into step 39 for tagmentation. During tagmentation, the DNA is cleaved and the transposase adapter simultaneously added to prepare the DNA ends for the attachment of the rapid adapter. The DNA is then cleaned using Precipitation Buffer (PTB) and incubated overnight.

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

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 sacrificial and will remain stuck 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.

MEDIDA OPCIONAL

At this point, the sample can be stored overnight at room temperature.

The next steps for DNA quantification are optional. Continue to the next stage of the protocol if quantification is to be omitted.

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.

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.

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.

FIN DEL PROCESO

During the Ultra-long DNA experiment incubation, complete the first wash and reload of the Duplex experiment.

13. Day 2: Washing and reloading Duplex library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
Sequencing Buffer (SB)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Library Solution (LIS)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 2: Duplex experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

For the Duplex experiment, up to four libraries prepared using the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) can be loaded on the PromethION Flow Cell during a sequencing run. We recommend washing your flow cell when ~20-25% of active pores are remaining, which typically occurs after ~20-24 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare the flow cell for loading a new library for further sequencing.

Navigate to the Pore Activity or the Pore Scan Results plot to see pore availability. Below is an example of pore states observed on a flow cell before and after wash steps are performed. The red asterisks indicates the reloads.

We recommend reloading your duplex experiment on days 2, 3 and 4 of the protocol.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

14. Day 3: Pore-C experiment

Material

Custom SPRI bead suspension

Consumibles

Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
Chilled phenol:chloroform:isoamyl alcohol in a 25:24:1 ratio, saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA (Sigma, P3803-400ML)
5 M NaCl (Sigma, 71386)
3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
Ethanol, 100% (e.g. Fisher, 16606002)
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
Tubos de ensayo Qubit™ (Invitrogen Q32856)
5 ml centrifuge tubes
2 ml Eppendorf DNA LoBind tubes

Instrumental

Temperature-controlled centrifuge
Mezclador Hula (mezclador giratorio suave)
Gradilla magnética
Thermal cycler or heat block
Fluorímetro Qubit (o equivalente para el control de calidad)
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P100
Pipeta y puntas P20
-80°C freezer storage

Day 3: Pore-C experiment overview

On the third day of the Pore-C experiment, the chimeric Pore-C dsDNA polymers are purified from the solution of polypeptide fragments and residual reaction buffers. The peptides are removed by using a phenol:chloroform extraction, followed by an ethanol precipitation to purify the DNA from the residual reaction buffers and phenol. The final Pore-C DNA extract is a pool of chimeric dsDNA polymers made of multiple ligated monomers which are sequenced to determine DNA interactions, proximity in sequence space and the three-dimensional structures of chromatin within the nucleus.

If the protein degradation has been successful, the interphase layer will be very thin and clear.

ATENCIÓN

Do not remove the interphase layer in the next step.

The solution will likely turn cloudy and then become clear once again.

For example, for a recovered volume of 2,000 µl, add:

40 µl of 5 M NaCl
200 µl of 3 M sodium acetate

The solution will likely turn cloudy and then become clear once again.

Note: If a –80°C freezer is not available or a pause in the protocol is required, an overnight incubation at –20°C can be used instead.

After the DNA pellets have dried, they may loosen from the tube.

CHECKPOINT

Quantify DNA concentration by using the Qubit dsDNA HS Assay Kit. Ensure a 1/10 dilution is used, as the Qubit reading will be affected by high salt concentration.

Note: The expected yield is ~7 μg per 10 million cells input for cell culture. Yields for other sample types may be reduced.

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

You can expect a 50-55% loss of DNA depending on a fragment length distribution of input material: the greater the proportion of short fragments (<1.5-2 kb), the greater the sample loss.

FIN DEL PROCESO

Store the sample at 4°C and complete the Ultra-long DNA experiment.

The Pore-C sample will undergo library preparation during day 4.

15. Day 3: Ultra-long DNA experiment

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Pipeta y puntas P1000
Pipeta y puntas P100
P200 pipette and tips
Pipeta y puntas P20
Wide-bore pipette tips

Day 3: Ultra-long DNA experiment overview

The eluate from the overnight incubation is removed and stored on ice until it is ready for loading onto the PromethION Flow Cell.

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.

16. Day 3: Priming and loading ultra-long DNA library on the PromethION Flow Cell

Material

Flow Cell Flush (FCF)
Flush Tether UL (FTU)
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

Consumibles

PromethION Flow Cell (FLO-PRO114M)
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Dispositivo PromethION 24/48
PromethION Flow Cell Light Shield
Pipeta y puntas P1000
Pipeta y puntas P200
Pipeta y puntas P20
Wide-bore pipette tips

Day 3: Ultra-long DNA experiment flow cell loading

Once the ultra-long DNA library has been prepared, the PromethION Flow Cell can be primed and the library prepared with the final sequencing reagents before loading for sequencing to begin. Due to the viscosity of the library, the flow cell priming and loading steps have been modified.

We recommend monitoring your sequencing run and to reload your flow cell when recommended to in the "Washing and reloading the PromethION Flow Cell with ultra-long DNA library" section. The Ultra-long DNA experiment can be reloaded up to three times, across days 4 and 5.

Reagent	Volume per flow cell
Sequencing Buffer UL (SBU)	100 µl
Loading Solution UL (LSU)	10 µl
DNA library	90 µl
Total	200 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.

Reagent	Volume
Flush Tether UL (FTU)	30 µl
Flow Cell Flush (FCF)	1170 µl
Total	1200 µl

IMPORTANTE

Una vez sacadas de la nevera, esperar 20 minutos antes de insertar las celdas de flujo en el dispositivo y así darles tiempo a que estén a temperatura ambiente. En entornos húmedos se puede formar condensación. Inspeccione las clavijas doradas del conector, situadas en la parte superior e inferior de la celda de flujo, en busca de condensación y si la hubiera, límpiela con una toallita sin pelusa. Procure que la almohadilla térmica (color gris oscuro) esté enganchada en la parte posterior.

Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

IMPORTANTE

Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Check that no air bubbles have been introduced to the inlet port during flow cell priming. If air is present, draw back a small volume to remove any air bubbles by using a P1000 pipette set to 200 µl and turning the pipette wheel (as per the instructions above).

There can be a delay between depressing the pipette and the library dispensing from the pipette tip. Dispense the library slowly, allowing the library to dispense from the pipette tip before depressing the pipette further.

It is important to dispense the library slowly to prevent air being introduced onto the flow cell.

Note: The DNA library loaded in this step is viscous and may not readily flow through the inlet port into the flow cell. In this case, we recommend applying negative pressure in the flow cell as explained in the steps below.

This step is required if the DNA library has not been fully absorbed into the inlet 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.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

FIN DEL PROCESO

Close the PromethION lid when ready to start a sequencing run on MinKNOW.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

17. Day 3: Washing and reloading Duplex library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 3: Duplex experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

We recommend reloading your duplex experiment on days 2, 3 and 4 of the protocol.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

18. Day 4: Pore-C experiment

Material

100-200 fmol Pore-C DNA extract
Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)

Consumibles

NEBNext FFPE DNA Repair Mix (NEB M6630)
NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
Qubit dsDNA HS Assay Kit (Invitrogen Q32851) (kit de ensayo ADNbc alta sensibilidad)
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)
Etanol al 80 % recién preparado con agua sin nucleasas
Tubos de ensayo Qubit™ (Invitrogen Q32856)
Tubos de 1,5 ml Eppendorf DNA LoBind
0.2 ml thin-walled PCR tubes

Instrumental

P1000 pipette and tips
Pipeta y puntas P100
Pipeta y puntas P10
Termociclador
Microfuge
Mezclador Hula (mezclador giratorio suave)
Gradilla magnética
Vortex mixer
Cubeta con hielo
Qubit fluorometer (or equivalent)

Day 4: Pore-C experiment overview

The Pore-C DNA extracts are prepared for sequencing using the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL), as follows: the extracted DNA is repaired and the ends prepared for adapter attachment using the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair/dA-tailing Module reagents. The sequencing adapters are attached to the DNA fragment ends before a clean-up step in preparation for sequencing on the PromethION Flow Cell.

CONSEJO

Se recomienda usar DNA Control Sample (DCS) en la preparación de la biblioteca para localizar y solucionar problemas. De todos modos, se puede omitir este paso y compensar el 1 µl extra con la muestra de ADN.

Para obtener un rendimiento óptimo, NEB recomienda lo siguiente:

Descongelar todos los reactivos en hielo.
Golpear suavemente los tubos de reactivos con el índice o invertirlos, para asegurarse de que estén bien mezclados.
Nota: No mezclar en vórtex las mezclas FFPE DNA Repair Mix, ni Ultra II End Prep Enzyme Mix.
Centrifugar los tubos antes de abrirlos.
Los tampones Ultra II End Prep Buffer y FFPE DNA Repair Buffer pueden tener un poco de precipitado. Dejar que la mezcla alcance la temperatura ambiente y mezclar pipeteando varias veces para romper el precipitado; para solubilizarlo, agitar el tubo en vórtex durante 30 s.
Nota: Es importante mezclar bien los tampones mediante vórtex.
El tampón FFPE DNA Repair Buffer puede tener un matiz amarillo; no importa si está así; se puede utilizar.

Transfer 100-200 fmol input DNA into a 1.5 ml Eppendorf DNA LoBind tube.
Adjust the volume to 47 μl with nuclease-free water.
Mix thoroughly by pipetting up and down, or by flicking the tube.
Spin down briefly in a microfuge

Entre cada adición, mezclar con la pipeta de 10 a 20 veces.

Reactivo	Volumen
ADN del paso anterior	47 µl
(opcional) DNA Control Sample (DCS)	1 µl
NEBNext FFPE DNA Repair Buffer	3,5 µl
NEBNext FFPE DNA Repair Mix	2 µl
Ultra II End-prep Reaction Buffer	3,5 µl
Ultra II End-prep Enzyme Mix	3 µl
Total	60 µl

CHECKPOINT

Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit.

MEDIDA OPCIONAL

If a pause is required, the sample can be stored overnight at 4°C.

IMPORTANTE

Aunque la ligasa recomendada de otros fabricantes se suministra con su propio tampón, la eficiencia del adaptador, Ligation Adapter (LA), es mayor cuando se usa el tampón Ligation Buffer (LNB) suministrado en el kit Ligation Sequencing Kit.

Entre cada adición, mezclar con la pipeta de 10 a 20 veces.

Reactivo	Volumen
Muestra de ADN del paso anterior	60 µl
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Ligation Adapter (LA)	5 µl
Total	100 µl

Dispose of the pelleted beads

CHECKPOINT

Cuantificar 1 μl de muestra eluida utilizando un fluorímetro Qubit.

IMPORTANTE

We recommend loading 10-20 fmol of this final prepared library onto the R10.4.1 flow cell.

Loading more than 20 fmol of DNA can reduce the rate of duplex read capture. Dilute the library in Elution Buffer if required.

FIN DEL PROCESO

The prepared library is used for loading onto the flow cell. Store the library on ice until ready to load.

19. Day 4: Priming and loading Pore-C library on the PromethION Flow Cell

Material

Sequencing Buffer (SB)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Library Solution (LIS)
Flow Cell Tether (FCT) (anclaje de celda de flujo)
Flow Cell Flush (FCF)

Consumibles

PromethION Flow Cell (FLO-PRO114M)
Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

Dispositivo PromethION 24/48
PromethION Flow Cell Light Shield
P1000 pipette and tips
P200 pipette and tips
Pipeta y puntas P20

Day 4: Pore-C experiment flow cell loading

Once the Pore-C DNA extracts have been prepared using the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL), the PromethION Flow Cell can be primed and the library prepared with the final sequencing reagents for the first library load to be sequenced.

We recommend monitoring your sequencing run and to reload your flow cell when recommended to in the "Washing and reloading Pore-C library on the PromethION Flow Cell" section. Pore-C DNA extracts are more prone to cause flow cell blocking and may require washing and reloading earlier during a sequencing run compared to the duplex condition. The Pore-C condition can be reloaded three times, across days 5, 6 and 7.

Uso de Library Solution (LIS)

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Una vez sacadas de la nevera, esperar 20 minutos antes de insertar las celdas de flujo en el dispositivo y así darles tiempo a que estén a temperatura ambiente. En entornos húmedos se puede formar condensación. Inspeccione las clavijas doradas del conector, situadas en la parte superior e inferior de la celda de flujo, en busca de condensación y si la hubiera, límpiela con una toallita sin pelusa. Procure que la almohadilla térmica (color gris oscuro) esté enganchada en la parte posterior.

Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

IMPORTANTE

Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

FIN DEL PROCESO

Close the PromethION lid when ready to start a sequencing run on MinKNOW.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

20. Day 4: Washing and reloading Duplex library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
Sequencing Buffer (SB)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Library Solution (LIS)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 4: Duplex experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

We recommend reloading your Duplex experiment on days 2, 3 and 4 of the protocol.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

After the final duplex library has been loaded and sequenced, the flow cell can be flushed with deionised water and returned to Oxford Nanopore. For further information, see the "Ending the experiment" step.

21. Day 4: Washing and reloading the PromethION Flow Cell with ultra-long DNA library

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Flush Tether UL (FTU)
Flow Cell Flush (FCF)
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

Consumibles

1.5 ml Eppendorf DNA LoBind tubes

Instrumental

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

Day 4: Ultra-long DNA experiment flow cell washing and reloading

We recommend reloading your PromethION Flow Cell with a fresh ultra-long DNA library to maintain high output, using the modified method for reloading a viscous library.

For the Ultra-long DNA experiment, up to three libraries prepared using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) can be loaded on the PromethION Flow Cell during a sequencing run. We recommend washing the flow cell when ~20-25% of active pores are remaining, which typically occurs after ~20-24 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare the flow cell for loading a new library for further sequencing.

Due to the viscosity of the library, the flow cell washing and reloading steps have been modified. It is also recommended to remove the waste fluid before washing the flow cell and before reloading of an ultra-long DNA library after each priming step.

We recommend washing and reloading on days 4 and 5 of this protocol.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

The waste fluid can be aspirated from either one of the ports, labelled 2 and 3 on the flow cell.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

It may be necessary to use a laboratory wipe to mop up any excess fluid that escapes from the flow cell waste ports 2 and 3.

The waste fluid can be aspirated from either one of the ports, labelled 2 and 3 on the flow cell.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagent	Volume per flow cell
Sequencing Buffer UL (SBU)	100 µl
Loading Solution UL (LSU)	10 µl
DNA library	90 µl
Total	200 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.

Reagent	Volume
Flush Tether UL (FTU)	30 µl
Flow Cell Flush (FCF)	1170 µl
Total	1200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

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

The waste liquid can be aspirated from either one of the ports, labelled 2 and 3.

It is important to dispense the library slowly to prevent air being introduced onto the flow cell.

This step is required if the DNA library has not been fully absorbed into the inlet 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.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

FIN DEL PROCESO

Resume the sequencing run on MinKNOW to continue data acquisition.

22. Day 5: Washing and reloading Pore-C library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
Sequencing Buffer (SB)
Library Solution (LIS)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 5: Pore-C experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

For the Pore-C experiment, up to four libraries prepared using the Pore-C protocol and Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) can be loaded on the PromethION Flow Cell during a sequencing run. We recommend washing your flow cell when ~20-25% of active pores are remaining, which typically occurs after ~18 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare a flow cell for loading a fresh library for further sequencing. Pore-C DNA extracts are prone to blocking and may require closer monitoring and washing earlier into the sequencing run than the duplex experiment to optimise sequencing output.

We recommend reloading your Pore-C experiment on days 5, 6 and 7.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

23. Day 5: Washing and reloading the PromethION Flow Cell with ultra-long DNA library

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Flush Tether UL (FTU)
Flow Cell Flush (FCF)
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

Consumibles

1.5 ml Eppendorf DNA LoBind tubes

Instrumental

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

Day 5: Ultra-long DNA experiment flow cell washing and reloading

We recommend reloading your PromethION Flow Cell with a fresh ultra-long DNA library to maintain high output, using the modified method for reloading a viscous library.

For the Ultra-long DNA experiment, up to three libraries prepared using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) can be loaded on the PromethION flow cell during a sequencing run. We recommend washing the flow cell when ~20-25% of active pores are remaining, which typically occurs after ~20-24 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare the flow cell for loading a new library for further sequencing.

We recommend washing and reloading on days 4 and 5 of this protocol.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagent	Volume per flow cell
Sequencing Buffer UL (SBU)	100 µl
Loading Solution UL (LSU)	10 µl
DNA library	90 µl
Total	200 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.

Reagent	Volume
Flush Tether UL (FTU)	30 µl
Flow Cell Flush (FCF)	1170 µl
Total	1200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

The waste liquid can be aspirated from either one of the ports, labelled 2 and 3.

It is important to dispense the library slowly to prevent air being introduced onto the flow cell.

This step is required if the DNA library has not been fully absorbed into the inlet 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.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

After the final ultra-long DNA library has been loaded and sequenced, the flow cell can be flushed with deionised water and returned to Oxford Nanopore. For further information, see the "Ending the experiment" step.

24. Day 6: Washing and reloading Pore-C library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
Sequencing Buffer (SB)
Library Solution (LIS)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Flow Cell Flush (FCF)
Flow Cell Tether (FCT)

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 6: Pore-C experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

For the Pore-C experiment, up to four libraries prepared using the Pore-C protocol and Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) can be loaded on the PromethION Flow Cell during a sequencing run. We recommend washing your flow cell when ~20-25% of active pores are remaining, which typical occurs after ~18 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare a flow cell for loading a fresh library for further sequencing. Pore-C DNA extracts are prone to blocking and may require closer monitoring and washing earlier into the sequencing run than the duplex experiment to optimise sequencing output.

We recommend reloading your Pore-C experiment on days 5, 6 and 7.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

25. Day 7: Washing and reloading Pore-C library on the PromethION Flow Cell

Material

Flow Cell Wash Kit XL (EXP-WSH004-XL)
Sequencing Auxiliary Vials V14 (EXP-AUX003)
Sequencing Buffer (SB)
Library Beads (LIB) (microesferas de carga de la biblioteca)
Library Solution (LIS)
Flow Cell Tether (FCT) (anclaje de celda de flujo)
Flow Cell Flush (FCF)

Consumibles

Tubos de 1,5 ml Eppendorf DNA LoBind

Instrumental

P1000 pipette and tips
Pipeta y puntas P20
Ice bucket with ice

Day 7: Pore-C experiment flow cell washing and reloading

We recommend washing and reloading your PromethION Flow Cell with a new library to maintain high data acquisition.

For the Pore-C experiment, up to four libraries prepared using the Pore-C protocol and Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) can be loaded on the PromethION flow cell during a sequencing run. We recommend washing your flow cell when ~20-25% of active pores are remaining, which typically occurs after ~18 hours of sequencing. Washing removes most of the initial library as well as unblocking pores to prepare a flow cell for loading a fresh library for further sequencing. Pore-C DNA extracts are prone to blocking and may require closer monitoring and washing earlier into the sequencing run than the duplex experiment to optimise sequencing output.

We recommend reloading your Pore-C experiment on days 5, 6 and 7.

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.

Reagent	Volume per flow cell
Wash Mix (WMX)	2 μl
Wash Diluent (DIL)	398 μl
Total	400 μl

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Close the inlet port.
Insert a P1000 pipette into a waste port and remove the waste buffer.

Note: As both the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette to 200 µl
Insert the tip into the inlet port
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.
Set a timer for a 5 minute incubation.

Using a P1000 pipette, take 200 µl of the flow cell wash mix
Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

The buffers used in this process are incompatible with conducting a Flow Cell Check step prior to loading the subsequent library. However, number of available pores will be reported after the next pore scan.

Reagents	Volume per flow cell
Flow Cell Flush (FCF)	1,170 µl
Flow Cell Tether (FCT)	30 µl
Total volume	1,200 µl

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

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

During this time, prepare the library for loading using the next steps in the protocol.

IMPORTANTE

Este vial contiene microesferas en suspensión. Las microesferas precipitan muy rápido; por eso, es fundamental mezclarlas justo antes de usar.

En la mayoría de experimentos de secuenciación, se recomienda usar Library Beads (LIB) . El reactivo Library Solution (LIS) está indicado para bibliotecas de ADN más viscosas.

Reagent	Volume per flow cell
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
Total	200 µl

Note: Library loading volume has been increased to improve array coverage.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Using a P1000 pipette, take 500 µl of the priming mix
Insert the pipette tip into the priming port, ensuring there are no bubbles in the tip
Slowly twist the pipette wheel down to load the flow cell (if possible with your pipette) or push down the plunger very slowly, leaving a small volume of buffer in the pipette tip.

IMPORTANTE

It is vital that the inlet port is closed before removing waste to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Ensure the inlet port is closed.
Insert a P1000 pipette into a waste port and remove the waste buffer

Note: As the inlet port is closed, no fluid should leave the sensor array area.

IMPORTANTE

Tenga cuidado a la hora de extraer el tampón de la celda de flujo. 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.

Set a P1000 pipette tip to 200 µl.
Insert the tip into the inlet port.
Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

IMPORTANTE

Para obtener resultados de secuenciación óptimos, coloque la pantalla protectora sobre la celda de flujo justo después de cargar la biblioteca.

Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

FIN DEL PROCESO

Resume the sequencing run in MinKNOW to continue data acquisition.

After the final Pore-C DNA library has been loaded and sequenced, the flow cell can be flushed with deionised water and returned to Oxford Nanopore. For further information, see the "Ending the experiment" step.

26. Duplex experiment: Data acquisition and basecalling

How to start sequencing duplex data

The sequencing device control, data acquisition and real-time simplex basecalling are carried out by the MinKNOW software. After simplex data has been basecalled, duplex basecalling is performed on Dorado, which is detailed below the MinKNOW settings.

Ensure you are using the most recent software version of MinKNOW.

We recommend basecalling with the Fast basecaller in real-time using the PromethION 24 or 48 device. After simplex basecalling on MinKnow, the data can be rebasecalled using the super accurate (SUP) model when performing duplex basecalling on Dorado.

For detailed instructions for setting up the device and sequencing run, see the MinKNOW protocol section for "Starting a sequencing run with PromethION 24 and 48".

For further detailed instructions for simplex and duplex basecalling, see the Kit 14 sequencing and duplex basecalling document.

MinKNOW settings for the Duplex experiment

For the Duplex experiment, we recommend keeping the sequencing parameters to their default settings for simplex basecalling.

Below are the current recommendations:

Positions

Flow cell position: [User defined] Experiment name: [User defined] Flow cell type: FLO-PRO114M Sample ID: [User defined]

Kit selection:

Kit selection: Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)

Run options:

Run duration: 100 hours Minimum read length: 200 bp

Adaptive sampling

Enrich or deplete sequencing: Off Barcoding balance: Not applicable

Advanced options

Active channel selection: On Time between pore scans: 1.5 hours Reserve pores: On

Analysis:

Basecalling

Basecalling: On Modified bases: On with '5mC' selected Model: Fast basecalling

Barcoding

Barcoding: Disabled

Alignment

Reference sequence: Off Note: We do not currently recommend live alignment during sequencing as it can slow down system processing.

Output:

Output format

Raw reads: On .FAST5: Off .POD5: On .FASTQ: On .BAM: On

Filtering: On Qscore: 8 Read length: Unfiltered Read splitting: Enabled Override read splitting min score: On

Overview of performing duplex basecalling:

Set up sequencing parameters in MinKNOW to perform simplex basecalling as described in "Basecalling Kit 14 simplex data". a. Basecall using the Fast basecaller. b. Output .POD5 files.
Using Dorado, re-basecall your simplex data with the following command to output simplex and duplex reads using the super-accurate (SUP) basecalling model. Other basecalling models can be used, as listed on the Dorado Github page.

$ dorado duplex dna_r10.4.1_e8.2_400bps_sup@v4.1.0 pod5s/ > duplex.bam

Dorado is a high-performance basecaller which is used to perform duplex basecalling. For further information about Dorado, please see the Dorado Github page.

Note: When running Dorado, we recommend stopping other basecalling for the best performance by maximising memory available to Dorado. This can be stopped and restarted when Dorado has finished via the GUI on MinKNOW.

Guppy may also be used to duplex basecall.

27. Ultra-long DNA experiment: Data acquisition and basecalling

How to start sequencing ultra-long read data

The sequencing device control, data acquisition and real-time simplex basecalling are carried out by the MinKNOW software.

Ensure you are using the most recent software version of MinKNOW.

We recommend basecalling with the high accuracy (HAC) basecaller in real-time using the PromethION 24 or 48 device. After basecalling, the simplex data can be rebasecalled using the super accurate (SUP) model and aligned to a reference genome.

For detailed instructions for setting up the device and sequencing run, see the MinKNOW protocol section for "Starting a sequencing run with PromethION 24 and 48".

MinKNOW settings for the Ultra-long DNA experiment

For the Ultra-long DNA experiment, we recommend keeping the sequencing parameters to their default settings for simplex basecalling.

Below are the current recommendations:

Poisitions

Flow cell position: [User defined]

Experiment name: [User defined]

Flow cell type: FLO-PRO114M

Sample ID: [User defined]

Kit selection:

Kit selection: Ultra-Long DNA Sequencing Kit V14 (SQK-LSK114)

Sequencing speed: Default (400 bps)

Run options:

Run duration: 72 hours

Minimum read length: 1000 bp

Adaptive sampling

Enrich or deplete sequencing: Off Barcoding balance: Not applicable

Advanced options

Active channel selection: On Time between pore scans: 1.5 hours Reserve pores: On

Analysis:

Basecalling

Basecalling: On Modified bases: On with '5mC' selected Model: High-accuracy basecalling (HAC)

Barcoding

Barcoding: Disabled

Alignment

Reference sequence: Off Note: We do not currently recommend live alignment during sequencing as it can slow down system processing.

Output:

Output format

Raw reads: On .FAST5: On .POD5: Off .FASTQ: On .BAM: On

Filtering: On Qscore: 9 Read length: Unfiltered Read splitting: Enabled Override read splitting min score: On

28. Pore-C experiment: Data acquisition and basecalling

How to start sequencing Pore-C data

The sequencing device control, data acquisition and real-time simplex basecalling are carried out by the MinKNOW software.

Ensure you are using the most recent software version of MinKNOW.

We recommend basecalling using the high accuracy (HAC) basecaller in real-time using the PromethION 24 or 48 device. After basecalling, the simplex data can be rebasecalled using the super accurate (SUP) model and aligned to a reference genome.

For detailed instructions for setting up the device and sequencing run, see the MinKNOW protocol section for "Starting a sequencing run with PromethION 24 and 48".

MinKNOW settings for the Pore-C experiment

For the Pore-C experiment, we recommend keeping the sequencing parameters to their default settings for simplex basecalling.

Below are the current recommendations:

Poisitions

Flow cell position: [User defined]

Experiment name: [User defined]

Flow cell type: FLO-PRO114M

Sample ID: [User defined]

Kit selection:

Kit selection: Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)

Sequencing speed: Default (400 bps)

Run options:

Run duration: 72 hours

Minimum read length: 200 bp

Adaptive sampling

Enrich or deplete sequencing: Off Barcoding balance: Not applicable

Advanced options

Active channel selection: On Time between pore scans: 1.5 hours Reserve pores: On

Analysis:

Basecalling

Basecalling: On Modified bases: On with '5mC' selected Model: High-accuracy basecalling (HAC)

Barcoding

Barcoding: Disabled

Alignment

Reference sequence: Off Note: We do not currently recommend live alignment during sequencing as it can slow down system processing.

Output:

Output format

Raw reads: On .FAST5: On .POD5: Off .FASTQ: On .BAM: On

Filtering: On Qscore: 9 Read length: Unfiltered Read splitting: Enabled Override read splitting min score: On

29. Downstream analysis

Telomere-to-telomere assembly

We currently recommend users with bioinformatic experience to analyse their data using Verkko, a genome assembly pipeline specifically designed for large, eukaryotic genomes and incorporates duplex data (high accuracy reads) and ultra-long reads for assembly of a human chromosome. This workflow starts by building a multiplex de Bruijn graph based on the duplex data and uses the ultra-long reads to simplify the graph for assembly of a chromosome.

To provide functionality for the detection of Pore-C ligation junctions and the genomic assembly of fragments, we recommend using Pore-C-Snakemake and Pore-C tools which will be made available through our EPI2ME products.

We will be releasing an EPI2ME workflow combining the three datasets for more accessibilty for users of all experience. Further information will be released on the Community.

30. 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.

31. Problemas durante la extracción de ADN/ARN y la preparación de bibliotecas

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.

Baja calidad de la muestra

Observación	Posible causa	Comentarios y acciones recomendadas
Baja pureza del ADN (la lectura del Nanodrop para ADN OD 260/280 es <1,8 y OD 260/230 es <2,0-2,2)	El método de extracción de ADN no proporciona la pureza necesaria	Los efectos de los contaminantes se muestran en la página Contaminants. Pruebe con un método de extracción alternativo que no provoque el arrastre de contaminantes. Considere realizar un paso adicional de limpieza SPRI.
Baja integridad del ARN (número de integridad del ARN <9,5 RIN o la banda ARNr se muestra como una mancha en el gel).	El ARN se degradó durante la extracción	Probar un método de extracción de ARN diferente. Encontrará más información sobre RIN en la página RNA Integrity Number. Asimismo, dispone de información adicional en la página DNA/RNA Handling.
El ARN tiene una longitud de fragmento más corta de lo esperado	El ARN se degradó durante la extracción	Probar un método de extracción de ARN diferente. Encontrará más información sobre RIN en la página RNA Integrity Number. Asimismo, dispone de información adicional en la página DNA/RNA Handling. Cuando se trabaje con ARN, recomendamos que el espacio de trabajo y el instrumental de laboratorio estén libres de ribonucleasas.

Escasa recuperación de ADN tras la limpieza con microesferas magnéticas AMPure

Observación	Posible causa	Comentarios y acciones recomendadas
Escasa recuperación	Pérdida de ADN debido a una proporción de microesferas magnéticas AMPure por muestra inferior a lo previsto.	1. Las microesferas magnéticas AMPure precipitan con rapidez; antes de añadirlas a la muestra hay que asegurarse de que estén bien resuspendidas. 2. Si la proporción de microesferas por muestra es inferior a 0.4:1, los fragmentos de ADN, sean del tamaño que sean, se perderán durante la limpieza.
Escasa recuperación	Los fragmentos de ADN son más cortos de lo esperado	Cuanto menor sea la proporción de microesferas magnéticas AMPure por muestra, más rigurosa será la selección de fragmentos largos frente a los cortos. Determinar siempre la longitud de la muestra de ADN en un gel de agarosa u otros métodos de electroforesis en gel, y, a continuación, calcular la cantidad adecuada de microesferas magnéticas que se debe utilizar.
Escasa recuperación tras la preparación de extremos	El paso de lavado utilizó etanol a <70 %	Cuando se utilice etanol a <70 %, el ADN se eluirá de las microesferas magnéticas. Asegúrese de utilizar el porcentaje correcto.

32. Problemas durante el experimento de secuenciación

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.

Menos poros al inicio de la secuenciación que después de verificar la celda de flujo

Observación	Posible causa	Comentarios y acciones recomendadas
MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo	Se introdujo una burbuja de aire en la matriz de nanoporos	Tras comprobar el número de poros presente en la celda de flujo, es imprescindible quitar las burbujas que haya cerca del puerto de cebado. Si no se quitan, pueden desplazarse a la matriz de nanoporos y dañar de manera irreversible los nanoporos expuestos al aire. En este vídeo se muestran algunas buenas prácticas para evitar que esto ocurra.
MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo	La celda de flujo no está colocada correctamente	Detener el ciclo de secuenciación, quitar la celda de flujo del dispositivo e insertarla de nuevo. Comprobar que está firmemente asentada en el dispositivo y que ha alcanzado la temperatura deseada. Si procede, probar con una posición diferente del dispositivo (GriION/PromethION).
MinKNOW presentó al inicio de la secuenciación un número de poros inferior al indicado durante la comprobación de la celda de flujo	La presencia de contaminantes en la biblioteca ha dañado o bloqueado los poros	El número de poros resultante tras la comprobación de la celda de flujo se realiza usando el control de calidad de las moléculas de ADN presentes en el tampón de almacenamiento de la celda de flujo. Al inicio de la secuenciación, se utiliza la misma biblioteca para estimar el número de poros activos. Por este motivo, se estima que puede haber una variabilidad del 10 % en el número de poros detectados. Tener un número de poros considerablemente inferior al inicio de la secuenciación puede deberse a la presencia de contaminantes en la biblioteca que hayan dañado las membranas o bloqueado los poros. Para mejorar la pureza del material de entrada tal vez sea necesario usar métodos de purificación o extracción de ADN/ARN alternativos. Los efectos de los contaminantes están descritos en la página Contaminants. Se recomienda, probar con un método de extracción alternativo que no provoque el arrastre de contaminantes.

Error en el script de MinKNOW

Observación	Posible causa	Comentarios y acciones recomendadas
MinKNOW muestra el mensaje "Error en el script"		Reiniciar el ordenador y reiniciar MinKNOW. Si el problema continúa, reúna los archivos de registro MinKNOW log files y contacte con el servicio de asistencia técnica. Si no dispone de otro dispositivo de secuenciación, recomendamos que guarde la celda de flujo con la biblioteca cargada a 4 °C y contacte con el servicio de asistencia técnica para recibir recomendaciones de almacenamiento adicionales.

Ocupación de poro por debajo del 40 %

Observación	Posible causa	Comentarios y acciones recomendadas
Ocupación de poro <40 %	No se cargó suficiente cantidad de biblioteca en la celda de flujo	Procure cargar la biblioteca al volumen y la concentración adecuados, tal como se indica en el protocolo correspondiente. Cuantificar la biblioteca antes de cargarla y calcular fmoles con herramientas como la calculadora Biomath de Promega, (opción "dsDNA: μg to fmol").
Ocupación de poro próxima a 0	Se utilizó el kit Ligation Sequencing Kit y los adaptadores de secuenciación no se ligaron al ADN	En la fase de ligación del adaptador, utilice NEBNext Quick Ligation Module (E6056) y el tampón Ligation Buffer (LNB) de Oxford Nanopore Technologies, suministrado en el kit de secuenciación. Recuerde añadir la cantidad correcta de cada reactivo. Prepare una biblioteca de control con lambda para valorar la integridad de los reactivos de otros fabricantes.
Ocupación de poro próxima a 0	Se utilizó el kit Ligation Sequencing Kit y en la fase de lavado, después de la ligación del adaptador, se utilizó etanol en lugar de Long Fragment Buffer (LFB) o Short Fragment Buffer (SFB).	El etanol puede desnaturalizar la proteína motor en los adaptadores de secuenciación. Usar Long Fragment Buffer (LFB) o Short Fragment Buffer (SFB) después de la ligación de los adaptadores.
Ocupación de poro próxima a 0	No hay anclaje en la celda de flujo	Los anclajes se añaden durante el cebado de la celda de flujo (Flush Tether (FLT) para los kits 9, 10 y 11, y Flow Cell Tether (FCT) para el kit 14). Asegurarse de añadir un anclaje —Flush Tether (FLT) o Flow Cell Tether (FCT)— al tampón antes del cebado; el tampón utilizado es Flush Buffer (FB) para los kits 9, 10 y 11 y Flow cell Flush (FCF) para el Kit 14.

Longitud de lectura más corta de lo esperado

Observación	Posible causa	Comentarios y acciones recomendadas
Longitud de lectura más corta de lo esperado	Fragmentación no deseada de la muestra de ADN	La longitud de lectura refleja la longitud del fragmento de la muestra de ADN. La muestra de ADN se puede fragmentar durante la extracción de la preparación de la biblioteca. 1. Consulte la sección de buenas prácticas de los métodos de extracción en la página Extraction Methods de la comunidad Nanopore. 2. Visualizar la distribución de la longitud de los fragmentos de las muestras de ADN en un gel de agarosa antes de proceder a la preparación de la biblioteca. En la imagen superior, la muestra 1 contiene alto peso molecular, mientras que la muestra 2 se ha fragmentado. 3. Durante la preparación de la biblioteca, evitar pipetear y agitar en vórtex cuando se mezclen los reactivos. Dar suaves golpes con el dedo o invertir el vial es suficiente.

Gran proporción de poros no disponibles

Observación	Posible causa	Comentarios y acciones recomendadas
Gran proporción de poros no disponibles (se muestran en azul oscuro en el panel de canales y en el gráfico de actividad de poros) Conforme pasa el tiempo, el gráfico de actividad de poros de arriba muestra una proporción creciente de poros no disponibles.	Hay contaminantes presentes en la muestra	Algunos contaminantes se pueden eliminar de los poros mediante la función de desbloqueo incorporada en MinKNOW. Si funciona, el estado de los poros cambiará a "sequencing pores" (secuenciación de poros). Si la porción poros no disponibles se mantiene elevada o aumenta, pruebe una de las siguientes opciones: 1. Realizar un enjuague de nucleasa con el kit de lavado Flow Cell Wash Kit (EXP-WSH004) 2. Realizar varios ciclos de PCR para intentar diluir cualquier contaminante que pueda estar causando problemas.

Gran proporción de poros inactivos

Observación	Posible causa	Comentarios y acciones recomendadas
Gran proporción de poros inactivos/no disponibles (se muestran en azul claro en el panel de canales y en el gráfico de actividad de poros. Los poros o membranas están dañados de manera irreversible)	Se han introducido burbujas de aire en la celda de flujo	Las burbujas de aire introducidas durante el cebado de la celda y la carga de la biblioteca pueden dañar los poros de forma permanente. Para conocer las buenas prácticas de cebado y carga de la celda de flujo, ver el vídeo Priming and loading your flow cell
Gran proporción de poros inactivos/no disponibles	Ciertos compuestos copurificados con ADN	Compuestos conocidos, incluidos los polisacáridos, se asocian generalmente con el ADN genómico de las plantas. 1. Consulte la página Plant leaf DNA extraction method. 2. Limpiar usando el kit QIAGEN PowerClean Pro. 3. Realizar una amplificación del genoma completo con la muestra original de ADNg utilizando el kit QIAGEN REPLI-g.
Gran proporción de poros inactivos/no disponibles	Hay contaminantes presentes en la muestra	Los efectos de los contaminantes se muestran en la página Contaminants. Probar con un método de extracción alternativo que no provoque el arrastre de contaminantes.

Fluctuación de la temperatura

Observación	Posible causa	Comentarios y acciones recomendadas
Fluctuación de la temperatura	La celda de flujo ha perdido contacto con el dispositivo	Comprobar que una almohadilla térmica cubra la placa metálica de la parte posterior de la celda de flujo. Reinsertar la celda de flujo y presionar para asegurarse de que las clavijas del conector estén bien conectadas al dispositivo. Si el problema continúa, contacte con el servicio de asistencia técnica.

Error al intentar alcanzar la temperatura deseada

Observación	Posible causa	Comentarios y acciones recomendadas
MinKNOW muestra el mensaje "Error al intentar alcanzar la temperatura deseada"	El dispositivo ha sido colocado en un lugar a una temperatura ambiente inferior a la media o en un lugar con escasa ventilación (lo que provoca el sobrecalientamiento de las celdas de flujo).	MinKNOW tiene un tiempo predeterminado para que las celdas de flujo alcancen la temperatura fijada. Una vez acabado el tiempo, aparece un mensaje de error, pero el experimento de secuenciación continua. Secuenciar a una temperatura incorrecta puede llevar a una disminución en el rendimiento y a generar un índice de calidad Qscore menor. Corrija la ubicación del dispositivo, procure que esté a temperatura ambiente y tenga buena ventilación; a continuación, reinicie el proceso en MinKNOW. Para obtener más información sobre el control de temperatura de MinKNOW Mk 1B, consulte la sección de preguntas frecuentes, FAQ.

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Documentation

Nanopore Learning

Telomere-to-telomere sequencing (T2T) on PromethION (SQK-LSK114-XL and SQK-ULK114)

Introduction to the protocol

Reagent and sample preparation

Day 1: Library preparation

Day 2: Library preparation

Day 3: Library preparation

Day 4: Library preparation

Day 5: Wash and reload flow cells

Day 6: Wash and reload flow cells

Day 7: Wash and reload flow cells

Sequencing and data analysis

Resolución de problemas

Document versions

PromethION: Protocol

Telomere-to-telomere sequencing (T2T) on PromethION (SQK-LSK114-XL and SQK-ULK114) V T2T_9179_v114_revL_31Jul2024

Contents

Introduction to the protocol

Reagent and sample preparation

Day 1: Library preparation

Day 2: Library preparation

Day 3: Library preparation

Day 4: Library preparation

Day 5: Wash and reload flow cells

Day 6: Wash and reload flow cells

Day 7: Wash and reload flow cells

Sequencing and data analysis

Resolución de problemas

Descripción general

1. Overview of the protocol

IMPORTANTE

This is a Legacy protocol

Introduction to the protocol

Steps in the sequencing workflow

Library preparation

Sequencing and analysis

IMPORTANTE

Compatibility of this protocol

2. Equipment and consumables

Material

Consumibles

Instrumental

Equipo opcional

Approximately 15 ml of whole blood is required as input for the sample preparation steps.

We recommend preparing your samples and the custom SPRI bead suspension a day ahead of the experiments to ensure maximum use of time each day.

Reactivos de otros fabricantes

NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing

Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents

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

Kit format with improved precipitation stars that can be used:

Kit format where stars should not be used:

Flow Cell Wash Kit XL (EXP-WSH004-XL) contents

3. Computer requirements and software

PromethION 24/48 IT requirements

PromethION 2 Solo IT requirements

Check your PromethION flow cell

Programas informáticos para secuenciar por nanoporos

MinKNOW

EPI2ME (opcional)

4. Custom SPRI bead preparation for the Pore-C experiment

Consumibles

Instrumental

Custom SPRI bead suspension preparation for Pore-C extraction

Prepare a custom buffer in a 2 ml Eppendorf DNA LoBind tube as follows for use in step 7.

Transfer 1 ml of resuspended Agencourt AMPure XP beads into two 2 ml Eppendorf DNA LoBind tubes, so that each tube contains 1 ml.

Place the tubes on a magnetic rack to pellet the beads until the solution is clear and colourless. Pipette off and discard the supernatant.

Remove the tubes from the magnet and resuspend the pellets with 1 ml of nuclease-free water. Pellet the beads on the magnet until supernatant is clear and colourless and pipette off the supernatant.

Repeat the previous step.

Spin down and place the tubes back on the magnet to pipette off any residual water.

Resuspend both tubes of pelleted beads in 200 µl of custom buffer and then pool both tubes into a single tube to a total of 400 µl.

Transfer the remaining custom buffer into the tube containing the pooled beads.

FIN DEL PROCESO

Store the beads at 4°C. Before use, bring the suspension to room temperature.

5. Whole blood sample preparation for the Pore-C experiment

Material

Consumibles

Instrumental