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

概要

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

重要

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.

重要

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

材料

>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)

消耗品

PromethION Flow Cell (FLO-PRO114M)
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
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). Alternatively, you can use the NEBNext® products below:
NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
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
Qubit™ Assay Tubes (Invitrogen, Q32856)
Ziplock bags
0.2 µm filter

装置

PromethION sequencing device
P1000 pipette and tips
P200 pipette and tips
P100 pipette and tips
P10 ピペットとチップ
P20 pipette and tips
Wide-bore pipette tips
Pasteur pipettes
Thermal cycler or heat block
Hula mixer（緩やかに回転するミキサー）
Magnetic rack
Vortex mixer
Temperature-controlled centrifuge
Microfuge
Ice bucket with ice
Thermomixer
Qubit蛍光光度計（またはQCチェックのための同等品）
Class I hood with active charcoal filter
-80°C freezer storage

オプション装置

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.

サードパーティー試薬

このプロトコールで使用されているすべてのサードパーティー試薬は、当社が検証し、使用を推奨しているものです。Oxford Nanopore Technologiesでは、それ以外の試薬を用いたテストは行っていません。

すべてのサードパーティ製試薬については、製造元の指示に従って使用の準備をすることをお勧めします。

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.

Software for nanopore sequencing

MinKNOW

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

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

EPI2ME (optional)

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

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

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.

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

消耗品

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)
nuclease-free waterで調整した 80% エタノール溶液
2 ml Eppendorf DNA LoBind tubes

装置

Magnetic rack
Hula mixer（緩やかに回転するミキサー）
Thermal cycler or heat block
P1000 pipette and tips
P200 ピペットとチップ
P100 pipette and tips
P10 ピペットとチップ
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.

最終ステップ

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

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

材料

5–10 ml whole blood

消耗品

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

装置

Pasteur pipettes
Temperature-controlled centrifuge with rotor for 2 ml and 50 ml tubes
Ice bucket with ice
P1000 pipette and tips
P200 pipette and tips
P100 pipette and tips
P20 pipette and tips
P10 ピペットとチップ

オプション装置

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

オプショナルステップ

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.

最終ステップ

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

材料

3 ml of whole blood
Custom SPRI bead suspension

消耗品

Puregene Blood Kit (QIAGEN, 158023)
ヌクレアーゼフリー水で用事調整した 80% エタノール溶液
Isopropanol, 100% (Fisher, 10723124)
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
1.5 ml Eppendorf DNA LoBind tubes
15 ml Falcon tubes
Qubit™ Assay Tubes (Invitrogen, Q32856)
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)

装置

Centrifuge and rotor suitable for 15 ml Falcon tubes
Heating block
ボルテックスミキサー
Qubit蛍光光度計（またはQCチェックのための同等品）

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.

最終ステップ

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

材料

1.6 ml of whole blood

消耗品

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

装置

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

ヒント

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

最終ステップ

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

材料

10 million white blood cells isolated from whole blood

消耗品

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
1.5 ml Eppendorf DNA LoBind tubes

装置

Class I hood with active charcoal filter
Temperature-controlled centrifuge
Thermal cycler or heat block
Thermomixer
Vortex mixer
Hula mixer（緩やかに回転するミキサー）
Ice bucket with ice
P1000 pipette and tips
P100 pipette and tips
P200 pipette and tips
P20 pipette and tips
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.

注意

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.

重要

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.

ヒント

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

重要

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.

オプショナルステップ

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.

重要

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

重要

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.

最終ステップ

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

9. Day 1: Duplex experiment

材料

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

消耗品

NEBNext FFPE DNA Repair Mix (NEB, M6630)
NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)
ヌクレアーゼフリー水で用事調整した 80% エタノール溶液
Qubit™ Assay Tubes (Invitrogen, Q32856)
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml 薄壁のPCRチューブ

装置

P1000 ピペット及びチップ
P100 ピペットとチップ
P20 pipette and tips
P10 ピペットとチップ
小型遠心機
Hula mixer（緩やかに回転するミキサー）
マグネットラック
アイスバケツ（氷入り）
ボルテックスミキサー
サーマルサイクラー
Qubit蛍光光度計（またはQCチェックのための同等品）

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.

ヒント

トラブルシューティングの為、DNAコントロールサンプル（DCS）を自身のライブラリーに添加して、プレップコントロール用に使用することを推奨します。ただし、このステップを省略して、1 µlをサンプルDNAで補うこともできます。

For optimal performance, NEB recommend the following:

Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.
The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

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

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA from the previous step	47 µl
DNA CS (optional)	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

Qubit蛍光光度計を使用して、溶出したサンプル1 µlを定量します。

オプショナルステップ

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

重要

推奨の他社製リガーゼ（ligase）には専用のバッファーが付属していますが、Ligation Sequencing Kitに付属のLigation Buffer (LNB)を使用した方が、Ligation Adapter (LA)のライゲーション効率が高くなります。

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA sample from the previous step	60 µl
Ligation Adapter (LA)	5 µl
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Total	100 µl

Dispose of the pelleted beads

CHECKPOINT

Qubit蛍光光度計を使用して、溶出したサンプル1 µlを定量します。

重要

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.

最終ステップ

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

材料

Sequencing Buffer (SB)
Library Beads (LIB)
Library Solution (LIS)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

消耗品

PromethION Flow Cell (FLO-PRO114M)
1.5 ml Eppendorf DNA LoBind tubes

装置

PromethION sequencing device
PromethION Flow Cell Light Shield
P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips

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.

Using the Library Solution

For most sequencing experiments, use the Library Beads (LIB) for loading your library onto the flow cell. However, for viscous libraries it may be difficult to load with the beads and may be appropriate to load using the 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

重要

冷蔵庫からフローセルを取り出した後にフローセルが室温に戻るまで20分待ってからPromethIONに差し込んでください。湿度の高い環境ではフローセルに結露が生じることがあります。フローセルの上面と下面にある金色のコネクターピンに結露がないかを点検し、結露が確認された場合はリントフリーのウェットティッシュで拭き取ってください。フローセル下面にヒートパッド（黒いパッド）があることを確認してください。

フローセルとコネクターを水平および垂直に並べてから、所定の位置にスムーズに挿入してください。
フローセルをしっかりと押し下げ、ラッチがかみ合い、カチッと音がして所定の位置に収まることを確認します。

重要

フローセルを誤った角度で挿入すると、PromethIONのピンが損傷し、シーケンス結果に影響を及ぼす可能性があります。PromethIONのピンが損傷している場合は、support@nanoporetech.com までご連絡ください。

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

ほとんどのシーケンス実験にはLibrary Beads （LIB）の使用を推奨します。しかし、より粘性の高いライブラリーにはLibrary Solution（LIS）を使ってください。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

最終ステップ

MinKNOWでシーケンスランを開始する準備ができたら、PromethIONの蓋を閉めてください。

フローセルをPromethIONにロードした後、実験を開始する前に最低10分間待ちます。この待ち時間があることで、よりシーケンス出力が向上します。

11. Day 2: Pore-C experiment

消耗品

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)
1.5 ml Eppendorf DNA LoBind tubes
2 ml Eppendorf DNA LoBind tubes

装置

Thermomixer
P1000 pipette and tips
P200 pipette and tips
P100 pipette and tips
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.

重要

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

ヒント

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.

重要

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.

最終ステップ

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

12. Day 2: Ultra-long DNA experiment

材料

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

消耗品

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
1.5 ml Eppendorf DNA LoBind tubes

装置

サーマルサイクラー
Temperature-controlled centrifuge
Microfuge
Hula mixer（緩やかに回転するミキサー）
Vortex mixer
Qubit fluorometer (or equivalent)
P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips
Wide-bore pipette tips
Ice bucket with ice

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.

ヒント

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.

ヒント

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

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

ヒント

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

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.

重要

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

重要

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.

オプショナルステップ

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.

ヒント

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

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.

最終ステップ

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

Resume the sequencing run in MinKNOW to continue data acquisition.

14. Day 3: Pore-C experiment

材料

Custom SPRI bead suspension

消耗品

Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
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)
Qubit™ Assay Tubes (Invitrogen, Q32856)
5 ml centrifuge tubes
2 ml Eppendorf DNA LoBind tubes

装置

Temperature-controlled centrifuge
Hula mixer（緩やかに回転するミキサー）
Magnetic rack
Thermal cycler or heat block
Qubit蛍光光度計（またはQCチェックのための同等品）
P1000 pipette and tips
P200 pipette and tips
P100 pipette and tips
P20 pipette and tips
-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.

注意

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.

最終ステップ

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P100 pipette and tips
P200 pipette and tips
P20 pipette and tips
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.

最終ステップ

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

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

材料

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

消耗品

PromethION Flow Cell (FLO-PRO114M)
1.5 ml Eppendorf DNA LoBind tubes

装置

PromethION sequencing device
PromethION Flow Cell Light Shield
P1000 ピペット及びチップ
P200 ピペットとチップ
P20 ピペットとチップ
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

重要

冷蔵庫からフローセルを取り出した後にフローセルが室温に戻るまで20分待ってからPromethIONに差し込んでください。湿度の高い環境ではフローセルに結露が生じることがあります。フローセルの上面と下面にある金色のコネクターピンに結露がないかを点検し、結露が確認された場合はリントフリーのウェットティッシュで拭き取ってください。フローセル下面にヒートパッド（黒いパッド）があることを確認してください。

フローセルとコネクターを水平および垂直に並べてから、所定の位置にスムーズに挿入してください。
フローセルをしっかりと押し下げ、ラッチがかみ合い、カチッと音がして所定の位置に収まることを確認します。

重要

フローセルを誤った角度で挿入すると、PromethIONのピンが損傷し、シーケンス結果に影響を及ぼす可能性があります。PromethIONのピンが損傷している場合は、support@nanoporetech.com までご連絡ください。

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

最終ステップ

MinKNOWでシーケンスランを開始する準備ができたら、PromethIONの蓋を閉めてください。

フローセルをPromethIONにロードした後、実験を開始する前に最低10分間待ちます。この待ち時間があることで、よりシーケンス出力が向上します。

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

Resume the sequencing run in MinKNOW to continue data acquisition.

18. Day 4: Pore-C experiment

材料

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

消耗品

NEBNext FFPE DNA Repair Mix (NEB, M6630)
NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
NEBNext Quick Ligation Module (NEB, E6056)
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)
ヌクレアーゼフリー水で用事調整した 80% エタノール溶液
Qubit™ Assay Tubes (Invitrogen, Q32856)
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubes

装置

P1000 pipette and tips
P100 pipette and tips
P10 ピペットとチップ
サーマルサイクラー
Microfuge
Hula mixer（緩やかに回転するミキサー）
Magnetic rack
Vortex mixer
Ice bucket with ice
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.

ヒント

トラブルシューティングの為、DNAコントロールサンプル（DCS）を自身のライブラリーに添加して、プレップコントロール用に使用することを推奨します。ただし、このステップを省略して、1 µlをサンプルDNAで補うこともできます。

For optimal performance, NEB recommend the following:

Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.
The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

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

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA from the previous step	47 µl
DNA CS (optional)	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

Qubit蛍光光度計を使用して、溶出したサンプル1 µlを定量します。

オプショナルステップ

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

重要

推奨の他社製リガーゼ（ligase）には専用のバッファーが付属していますが、Ligation Sequencing Kitに付属のLigation Buffer (LNB)を使用した方が、Ligation Adapter (LA)のライゲーション効率が高くなります。

Between each addition, pipette mix 10-20 times.

Reagent	Volume
DNA sample from the previous step	60 µl
Ligation Adapter (LA)	5 µl
Ligation Buffer (LNB)	25 µl
NEBNext Quick T4 DNA Ligase	10 µl
Total	100 µl

Dispose of the pelleted beads

CHECKPOINT

Qubit蛍光光度計を使用して、溶出したサンプル1 µlを定量します。

重要

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.

最終ステップ

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

材料

Sequencing Buffer (SB)
Library Beads (LIB)
Library Solution (LIS)
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

消耗品

PromethION Flow Cell (FLO-PRO114M)
1.5 ml Eppendorf DNA LoBind tubes

装置

PromethION sequencing device
PromethION Flow Cell Light Shield
P1000 pipette and tips
P200 pipette and tips
P20 pipette and tips

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.

Using the Library Solution

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

重要

冷蔵庫からフローセルを取り出した後にフローセルが室温に戻るまで20分待ってからPromethIONに差し込んでください。湿度の高い環境ではフローセルに結露が生じることがあります。フローセルの上面と下面にある金色のコネクターピンに結露がないかを点検し、結露が確認された場合はリントフリーのウェットティッシュで拭き取ってください。フローセル下面にヒートパッド（黒いパッド）があることを確認してください。

フローセルとコネクターを水平および垂直に並べてから、所定の位置にスムーズに挿入してください。
フローセルをしっかりと押し下げ、ラッチがかみ合い、カチッと音がして所定の位置に収まることを確認します。

重要

フローセルを誤った角度で挿入すると、PromethIONのピンが損傷し、シーケンス結果に影響を及ぼす可能性があります。PromethIONのピンが損傷している場合は、support@nanoporetech.com までご連絡ください。

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

最終ステップ

MinKNOWでシーケンスランを開始する準備ができたら、PromethIONの蓋を閉めてください。

フローセルをPromethIONにロードした後、実験を開始する前に最低10分間待ちます。この待ち時間があることで、よりシーケンス出力が向上します。

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P200 ピペットとチップ
P20 ピペットとチップ

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.

ヒント

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

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

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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.

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

最終ステップ

Resume the sequencing run on MinKNOW to continue data acquisition.

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P200 ピペットとチップ
P20 ピペットとチップ

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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

最終ステップ

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

材料

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

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

Resume the sequencing run in MinKNOW to continue data acquisition.

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

材料

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

消耗品

1.5 ml Eppendorf DNA LoBind tubes

装置

P1000 pipette and tips
P20 pipette and tips
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.

ヒント

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

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

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

重要

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.

重要

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

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.

重要

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.

重要

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

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

Library Beads（LIB）チューブにはビーズの懸濁液が入っています。これらのビーズはすぐに沈殿するので、使用直前に混合することが重要です。

試薬	フローセルあたりの容量
Sequencing Buffer (SB)	100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS)	68 µl
DNA library	32 µl
合計	200 µl

注）アレイカバレッジを向上させるため、ライブラリーのローディング量を増やしました。

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

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.

重要

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.

重要

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

P1000ピペットチップを200µlにセットします。
チップをインレットポートに挿入します。
ダイヤルが220～230µlを示すまで、またはピペットチップに少量のバッファーが入るのが確認できるまで、ホイールを回します。

重要

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

ライトシールドとインレットポートをフローセルのインレットポートカバーに合わせます。ライトシールドの前縁をフローセルIDの上に位置するようにします。
ライトシールドをインレットポートカバーの周囲にしっかりと押し付けます。インレットポートクリップがインレットポートカバーの下にカチッとはまるようになっています。

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.

最終ステップ

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. フローセルの再利用と返却

材料

Flow Cell Wash Kit (EXP-WSH004)

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

ヒント

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

フローセルの返却方法は hereをご覧ください。

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

重要

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

31. DNA/RNA抽出、およびライブラリ調製時の問題点

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

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

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

サンプルの品質が低い

問題点	この問題が生じた可能性のある原因	解決策とコメント
DNAの純度が低い（DNAのOD 260/280のナノドロップ測定値が1.8未満およびOD 260/230が2.0～2.2未満）	DNA抽出で必要な純度が得られていない	夾雑物の影響は、 Contaminants に示されています。コンタミネーションをもたらさないために別の抽出方法extraction method をお試しください。. 追加のSPRIクリーンアップステップの実施を検討して下さい。
低いRNA インテグリティー（RNA Integrity Number: <9.5 RIN、またはrRNAバンドがゲル上でスメアになっている）	抽出中にRNAが分解された	別のRNA抽出方法 RNA extraction methodを試してください。RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、 DNA/RNA Handling のページをご覧ください。
RNAのフラグメントが予想より短い	抽出中にRNAが分解された	別のRNA抽出方法 RNA extraction methodを試してください。 RINの詳細については、 RNA Integrity Number の資料を参照してください。詳細については、DNA/RNA Handling のページをご覧ください。 RNAを扱う際には、RNaseフリーの環境で作業し、実験器具もRNaseフリーにしておくことをお勧めします。

AMPureビーズクリーンアップ後のDNA回収率が低い

問題点	この問題が生じた可能性のある原因	解決策とコメント
低回収率	AMPureビーズとサンプルの比率が予想していたのよりも低いことによるDNAの損失	1. AMPureビーズはすぐに沈降するため、サンプルに添加する前によく再懸濁させてください。 2. AMPureビーズ対サンプル比が0.4:1未満の場合、どのようなサイズのDNA断片でもクリーンアップ中に失われます。
低回収率	DNA断片が予想よりも短い	サンプルに対するAMPureビーズの比率が低いほど、短い断片に対する選択が厳しくなります。アガロースゲル(または他のゲル電気泳動法)上でインプットDNAの長さを設定してから、使用するAMPureビーズの適切な量を計算してください。
エンドプレップ後の収率が低い	洗浄ステップで使用したエタノール濃度が低い（70%未満）。	エタノールが70%未満の場合、DNAは洗浄中にビーズから溶出されます。必ず正しい濃度（％）のエタノールを使用してください。

32. シークエンス実行中の問題

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

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

シークエンス開始時のポアがフローセルチェック後よりも少ない場合

問題点	予想される原因	解決策とコメント
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。	ナノポアアレイに気泡が入ってしまった。	フローセルチェックをした後、フローセルをプライミングする前に、プライミングポート付近の気泡を取り除くことが必要です。気泡を取り除かないと、気泡がナノポアアレイに移動し、空気に触れたたナノポアが不可逆的なダメージを負った可能性がある。これを防ぐための最適な方法が、 this videoで紹介されています。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。	フローセルがデバイスに正しく挿入されていない。	シークエンスランを停止し、フローセルをシークエンス装置から取り出します。次に再度フローセルを挿入し、装置にしっかりと固定され、目標温度に達していることを確認します。GridIONやPromethIONの場合は別のフローセルの位置をお試しください。
MinKNOWのフローセルチェックで確認されたポアの数より、シークエンシング開始時のポア数が少なく表示された。	ライブラリー内の汚染物質がポアを失活させたり塞いだりしている。	フローセルチェックの際のポア数は、フローセル保存バッファー中のQC用のDNA分子を用いて計測されます。シークエンシングの開始時は、ライブラリ自体を使用してアクティブなポア数を推定します。このため、フローセルチェックとRun開始時のポア数は、約10％程度の変動が起こります。シークエンシング開始時に報告されたポアの数が大幅に減少している場合は、ライブラリー中の汚染物質がメンブレンを損傷していたり、ポアをブロックしている可能性があります。インプット材料の純度を向上させるために、別のDNA/RNA抽出または精製方法が必要となる場合があります。コンタミネーションの影響は、Contaminants Know-how pieceを参照にして下さい。夾雑物を除去するために別の抽出方法extraction method をお試しください。

MinKNOWのスクリプトに問題

問題点	この問題が生じた可能性のある原因	解決策とコメント
MinKNOW に「Script failed」と表示されている"		コンピューターを再起動し、MinKNOWを再起動します。問題が解決しない場合は MinKNOW log files MinKNOWログファイルを収集し、テクニカルサポートにご連絡ください。他のシークエンシングデバイスをお持ちでない場合は、フローセルとロードしたライブラリーを4℃で保管することをお勧めします。詳細な保管方法については、テクニカルサポートにお問い合わせください。

ポア占有率が40%未満

問題点	予想される原因	解決策とコメント
ポアの占有率が40%以下	フローセルに十分なライブラリーがロードされていなかった。	シークエンシングライブラリーを正確に濃度測定し、適切な容量がフローセルにロードされていることを確認してください(詳しくはそれぞれのプロトコールをご覧ください)。ロードする前にライブラリーを定量し、 Promega Biomath Calculatorなどのツールを使用してfmolを計算してください。[dsDNA: µ g to fmol]を選択してください。
ポア占有率が0に近い	Ligation Sequencing Kitを使用したが、シークエンシングアダプターはDNAにライゲーションしなかった。	シークエンシングアダプターのライゲーションステップでは、必ずNEBNext Quick Ligation Module（E6056）とOxford Nanopore Technologies Ligation Buffer（LNB、シークエンスキットに付属されています。）を使用し、各試薬の量を適切に使用してください。サードパーティ試薬の完全性をテストするために、Lambdaのコントロールライブラリーを調製することもできます。
ポア占有率が0に近い	シークエンシングアダプターライゲーション後の洗浄工程で、LFBまたはSFBの代わりにエタノールを使用してしまった。	エタノールはシークエンシングアダプター上のモータータンパク質を変性させる可能性があります。シークエンシングアダプターのライゲーション後にLFBまたはSFBバッファーを使用したことを確認して下さい。
ポア占有率が0に近い	フローセルにテザーがない	テザーはフローセルのプライミング時に追加されます（キット9、10、11はFLTチューブ、キット14はFTUを使用。ウルトラロングのDNAキットにはFTUを使用。）プライミングの前に、FLT/FCT/FTUがバッファー（キット9、10、11はFB、キット14はFCF）に添加されていることを確認してください。

予想より短いリード長

問題点	予想される原因	解決策とコメント
予想より短いリード長	DNAサンプルの不要な断片化	読み取り長はサンプルDNA断片の長さを反映します。サンプルDNAは、抽出およびライブラリー調製中の操作で断片化した可能性があります。 1. 抽出の最適な方法については、Extraction Methods の抽出方法を参照してください。 2. ライブラリー調製に進む前に、アガロースゲル電気泳動で、サンプルDNAのフラグメント長の分布を確認してください。上の画像では、サンプル1は高分子量ですが、サンプル2は断片化されています。 3. ライブラリー調製中は、試薬を混合するためのピペッティングやボルテックス操作は、プロトコルで指示がないかぎり行わないでください。

利用できないポアの割合が多い場合

問題点	予想される原因	解決策とコメント
利用できないポアの割合が大きい（チャンネルパネルとポアのアクティブポートで青く表示されています）上のアクティブなポアの図は、時間の経過とともに「利用できない」ポアの割合が増加していることを示しています。	サンプル内に不純物が含まれている	一部のポアに吸着する不純物は、MinKNOWに組み込まれたポアのブロック解除機能によって、ポアから除去することができます。このステップが完了すると、ポアの状態が「sequencing pore」に戻ります。利用できないポアの部分が多いか、増加した場合: 1.Flow Cell Wash Kit nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) を用いて、ヌクレアーゼ洗浄を行うことができます。又は 2. PCRを数サイクル実行してサンプルDNAの量を増やし、サンプルDNAに含まれる問題の不純物が相対的に減る（希釈される）ようにします。

Inactiveのポアの割合が高い

問題点	予想される原因	解決策とコメント
利用できない(inactive/unavailable)ポアの割合が高い（チャネルパネルとポアアクティブポートでは水色で表示されています）ポアまたは膜に損傷が起きてしまった。	気泡がフローセルに混入した。	フローセルのプライミングやライブラリーのロードで気泡が入ると、ポアに不可逆的なダメージを与える可能性があります。推奨の操作方法については、Priming and loading your flow cell のビデオをご覧ください。
利用できないポアの割合が多い場合	サンプルDNAに含まれる不純物	既知の化合物問題で、サンプルDNAに多糖類が含まれた事で、植物のゲノムDNAと結合しポアをブロックした。 1. 植物葉DNA抽出法 Plant leaf DNA extraction methodをご参照ください。 2. QIAGEN PowerClean Pro キットを使用してクリーンアップして下さい。 3. QIAGEN REPLI-g kit.キットを使用して、元のgDNAサンプルで全ゲノム増幅を実行します。
利用できないポアの割合が多い場合	サンプル内に不純物が含まれている	不純物の影響は、 Contaminants のノウハウを参照して下さい。サンプルDNAに不純物を残留させないために別の抽出方法をお試しください。

温度変動

問題点	予想される原因	解決策とコメント
温度変動	フローセルとデバイスの接続が途切れている。	フローセルの背面にある金属プレートを覆っているヒートパッドがあることを確認してください。フローセルを再度挿入し、コネクターピンがデバイスにしっかりと接触していることを確認するために軽く押してください。問題が解決しない場合は、テクニカルサービスにご連絡してください。

目標温度に到達しない場合

問題点	予想される原因	解決策とコメント
MinKNOWが "Failed to reach target temperature "(目標温度に達しなかった)と表示する。"	装置が通常の室温より低い場所、または風通しの悪い場所（排気が出来ない場所）に置かれた時にフローセルが過熱してします。	MinKNOWでは、フローセルが目標温度に到達するまでの既定の時間枠があります。時間枠を超えると、エラーメッセージが表示され、シークエンシング実験が続行されます。しかし、不適切な温度でシークエンスを行うと、スループットが低下し、qスコアが低下する可能性があります。シークエンシングデバイスが風通しの良い室温に置かれていることを確認して、MinKNOW再スタートしてください。MinION Mk 1Bの温度制御の詳細については、FAQ を参照してください。

Device:

London Calling 2025

すべての製品

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

トラブルシューティング

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

トラブルシューティング

概要

1. Overview of the protocol

重要

This is a Legacy protocol

Introduction to the protocol

Steps in the sequencing workflow

Library preparation

Sequencing and analysis

重要

Compatibility of this protocol

2. Equipment and consumables

材料

消耗品

装置

オプション装置

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.

サードパーティー試薬

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

Software for nanopore sequencing

MinKNOW

EPI2ME (optional)

Check your PromethION flow cell

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

消耗品

装置

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.

最終ステップ

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

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

材料

消耗品

装置