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

概览

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)

耗材

PromethION Flow Cell (FLO-PRO114M)
Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（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)
供Oxford Nanopore Technologies®连接测序使用的NEBNext®配套模块（目录号E7180S或 E7180L），或使用以下三种NEBNext®产品：
NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
NEBNext 快速连接模块（NEB，E6056）
NEBNext FFPE DNA 修复混合液（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 离心管
0.2 ml thin-walled PCR tubes
Qubit™ 分析管（Invitrogen, Q32856）
Ziplock bags
0.2 µm filter

仪器

PromethION测序设备
P1000 pipette and tips
P200 移液枪和枪头
P100移液枪和枪头
P10 移液枪和枪头
P20 移液枪和枪头
Wide-bore pipette tips
Pasteur pipettes
Thermal cycler or heat block
Hula混匀仪（低速旋转式混匀仪）
磁力架
Vortex mixer
Temperature-controlled centrifuge
迷你离心机
盛有冰的冰桶
Thermomixer
Qubit荧光计（或用于质控检测的等效仪器）
Class I hood with active charcoal filter
-80°C freezer storage

可选仪器

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.

第三方试剂

Oxford Nanopore Technologies推荐您使用本实验指南中提及的所有第三方试剂，并已对其加以验证。我们尚未对其它替代试剂进行测试。

我们建议您按制造商说明准备待用的第三方试剂.

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. 计算机要求及软件

PromethION 24/48 的IT配置要求

PromethION设备的硬件能够同时控制多达24个（适用于P24型号）或48个（适用于P48型号）测序实验，并采集数据。此外，设备借助高性能GPU技术，可以实时识别碱基。请参阅 PromethION IT 配置要求文档，了解更多信息。

PromethION 2 Solo 的IT配置要求

作为一款小型台式测序设备，PromethION 2 Solo可独立或同时运行两张测序芯片。您只需将PromethION 2 Solo连接到GridION Mk1或符合最低技术规格要求的独立计算机，即可实现数据的实时采集和分析。欲了解更多信息，请参阅PromethION 2 Solo IT 配置要求文档。

纳米孔测序相关软件

MinKNOW

MinKNOW软件负责控制纳米孔测序仪、实时收集测序数据并进行碱基识别。您将在每次测序实验中使用MinKNOW。MinKNOW还可以通过条形码标记序列，并在测序完成后对数据进行碱基/序列识别及拆分。

有关如何运行MinKNOW软件的说明，请参考 MinKNOW实验指南中的相关部分。

EPI2ME （可选）

EPI2ME云端平台为下机数据提供进一步的分析（如，与Lambda基因组比对、条形码拆分、或分类学鉴定）。EPI2ME 仅在您需要对下机数据进一步分析时使用。

有关如何创建EPI2ME账户并安装EPI2ME桌面代理（EPI2ME Desktop Agent）的说明，请参考EPI2ME平台使用指南。

测序芯片质检

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到PremethION测序芯片12周之内进行。Oxford Nanopore Technologies会对活性孔数量少于 5000 的芯片进行替换**。

** 请注意：自收到之日起，芯片须一直贮存于Oxford Nanopore Technologies推荐的条件下。且质检结果须在质检后的两天内递交给我们。请您按照测序芯片质检文档中的说明进行芯片质检。

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

耗材

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)
新制备的80%乙醇（用无核酸酶水配制）
2 ml Eppendorf DNA LoBind 离心管

仪器

磁力架
Hula混匀仪（低速旋转式混匀仪）
Thermal cycler or heat block
P1000 移液枪和枪头
P200 移液枪和枪头
P100 pipette and tips
P10 移液枪和枪头
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.

步骤结束

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

耗材

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 离心管

仪器

Pasteur pipettes
Temperature-controlled centrifuge with rotor for 2 ml and 50 ml tubes
盛有冰的冰桶
P1000 pipette and tips
P200 移液枪和枪头
P100移液枪和枪头
P20 移液枪和枪头
P10 移液枪和枪头

可选仪器

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

可选操作

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.

步骤结束

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

耗材

Puregene Blood Kit (QIAGEN, 158023)
新制备的80%乙醇（用无核酸酶水配制）
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 离心管
15 ml Falcon tubes
Qubit™ 分析管（Invitrogen, Q32856）
Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）

仪器

Centrifuge and rotor suitable for 15 ml Falcon tubes
Heating block
涡旋混匀仪
Qubit荧光计（或用于质控检测的等效仪器）

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.

步骤结束

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

耗材

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 离心管

仪器

Temperature-controlled microfuge
P1000 移液枪和枪头
P200 移液枪和枪头
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

提示

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

步骤结束

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

耗材

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 离心管
1.5 ml Eppendorf DNA LoBind 离心管

仪器

Class I hood with active charcoal filter
Temperature-controlled centrifuge
Thermal cycler or heat block
Thermomixer
Vortex mixer
Hula混匀仪（低速旋转式混匀仪）
盛有冰的冰桶
P1000 pipette and tips
P100移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头
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.

提示

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.

可选操作

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.

步骤结束

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

9. DNA修复和末端制备 (5)

材料

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

耗材

NEBNext FFPE DNA 修复混合液（NEB，M6630）
NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
NEBNext 快速连接模块（NEB，E6056）
Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
无核酸酶水（如ThermoFisher，AM9937）
新制备的80%乙醇（用无核酸酶水配制）
Qubit™ 分析管（Invitrogen, Q32856）
1.5 ml Eppendorf DNA LoBind 离心管
0.2 ml薄壁PCR管

仪器

P1000 移液枪和枪头
P100 移液枪和枪头
P20 移液枪和枪头
P10 移液枪和枪头
迷你离心机
Hula混匀仪（低速旋转式混匀仪）
磁力架
盛有冰的冰桶
涡旋混匀仪
热循环仪
Qubit荧光计（或用于质控检测的等效仪器）

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.

提示

我们推荐您在制备文库时，加入1μl 的DNA参照，以便日后进行问题排查。您也可以选择跳过此步骤，以额外的1μl DNA样本替代。

为获得最优表现，NEB建议如下：

于冰上解冻所有试剂。
轻弹并/或翻转各管，确保各试剂充分混匀。
注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。
同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。
Ultra II 末端修复缓冲液和 FFPE DNA 修复缓冲液内可能出现少量沉淀。待此两管液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后涡旋振荡30秒，以确保沉淀充分溶解。
注意： 请务必涡旋振荡混匀缓冲液。
FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

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

每添加一样试剂后，请吹打混匀10-20次，再添加下一样试剂。

试剂	体积
DNA 样本	47 µl
DNA参照（非必需）	1 µl
NEBNext FFPE修复缓冲液	3.5 µl
NEBNext FFPE修复混合液	2 µl
Ultra II 末端修复反应缓冲液	3.5 µl
Ultra II 末端修复酶混合物	3 µl
总体积	60 µl

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

可选操作

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

重要

尽管第三方连接酶产品可能也附带缓冲液，但使用连接测序试剂盒中提供的连接缓冲液（LNB）时，连接接头（LA）的连接效率会更高。

每添加一样试剂后，请吹打混匀10-20次，再添加下一样试剂。

试剂	体积
前一步骤所得DNA样品	60 µl
连接缓冲液（LNB）	25 µl
NEBNext快速T4 DNA连接酶	10 µl
连接接头（LA）	5 µl
总体积	100 µl

弃掉磁珠。

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

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.

步骤结束

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

测序缓冲液（SB）
文库颗粒（LIB）
文库溶液（LIS）
测序芯片系绳（FCT）
测序芯片冲洗液（FCF）

耗材

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

仪器

PromethION测序设备
PromethION 测序芯片遮光片
P1000 移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头

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.

使用文库溶液

对大多数测序实验，我们建议您使用文库颗粒（LIB）给测序芯片上样。然而，对于粘稠的文库，借助文库颗粒上样可能会比较困难，此时使用文库溶液（LIS）可能更为合适。

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

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

将测序芯片与连接器横竖对齐，以便顺利卡入。
用力下压芯片至卡槽，并确认卡夹位置归位。

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

11. Day 2: Pore-C experiment

耗材

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 离心管
2 ml Eppendorf DNA LoBind 离心管

仪器

Thermomixer
P1000 pipette and tips
P200 移液枪和枪头
P100移液枪和枪头
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.

提示

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.

步骤结束

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)

耗材

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 离心管
1.5 ml Eppendorf DNA LoBind 离心管

仪器

热循环仪
Temperature-controlled centrifuge
迷你离心机
Hula混匀仪（低速旋转式混匀仪）
Vortex mixer
Qubit荧光计（或用于质控检测的等效仪器）
P1000移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头
Wide-bore pipette tips
盛有冰的冰桶

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.

提示

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.

提示

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.

提示

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.

可选操作

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.

提示

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.

步骤结束

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)
测序缓冲液（SB）
文库颗粒（LIB）
文库溶液（LIS）
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

Resume the sequencing run in MinKNOW to continue data acquisition.

14. Day 3: Pore-C experiment

材料

Custom SPRI bead suspension

耗材

Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（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™ 分析管（Invitrogen, Q32856）
5 ml centrifuge tubes
2 ml Eppendorf DNA LoBind 离心管

仪器

Temperature-controlled centrifuge
Hula混匀仪（低速旋转式混匀仪）
磁力架
Thermal cycler or heat block
Qubit荧光计（或用于质控检测的等效仪器）
P1000 pipette and tips
P200 移液枪和枪头
P100移液枪和枪头
P20 移液枪和枪头
-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.

步骤结束

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

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000移液枪和枪头
P100移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头
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.

步骤结束

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)

耗材

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

仪器

PromethION测序设备
PromethION 测序芯片遮光片
P1000 移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头
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

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

将测序芯片与连接器横竖对齐，以便顺利卡入。
用力下压芯片至卡槽，并确认卡夹位置归位。

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

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)

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

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)

耗材

NEBNext FFPE DNA 修复混合液（NEB，M6630）
NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
NEBNext 快速连接模块（NEB，E6056）
Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
Agencourt AMPure XP Beads (Beckman Coulter™, A63881)
Nuclease-free water (e.g. ThermoFisher, AM9937)
新制备的80%乙醇（用无核酸酶水配制）
Qubit™ 分析管（Invitrogen, Q32856）
1.5 ml Eppendorf DNA LoBind 离心管
0.2 ml thin-walled PCR tubes

仪器

P1000 pipette and tips
P100移液枪和枪头
P10 移液枪和枪头
热循环仪
迷你离心机
Hula混匀仪（低速旋转式混匀仪）
磁力架
Vortex mixer
盛有冰的冰桶
Qubit荧光计（或用于质控检测的等效仪器）

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.

提示

我们推荐您在制备文库时，加入1μl 的DNA参照，以便日后进行问题排查。您也可以选择跳过此步骤，以额外的1μl DNA样本替代。

为获得最优表现，NEB建议如下：

于冰上解冻所有试剂。
轻弹并/或翻转各管，确保各试剂充分混匀。
注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。
同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。
Ultra II 末端修复缓冲液和 FFPE DNA 修复缓冲液内可能出现少量沉淀。待此两管液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后涡旋振荡30秒，以确保沉淀充分溶解。
注意： 请务必涡旋振荡混匀缓冲液。
FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。

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

每添加一样试剂后，请吹打混匀10-20次，再添加下一样试剂。

试剂	体积
DNA 样本	47 µl
DNA参照（非必需）	1 µl
NEBNext FFPE修复缓冲液	3.5 µl
NEBNext FFPE修复混合液	2 µl
Ultra II 末端修复反应缓冲液	3.5 µl
Ultra II 末端修复酶混合物	3 µl
总体积	60 µl

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

可选操作

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

重要

尽管第三方连接酶产品可能也附带缓冲液，但使用连接测序试剂盒中提供的连接缓冲液（LNB）时，连接接头（LA）的连接效率会更高。

每添加一样试剂后，请吹打混匀10-20次，再添加下一样试剂。

试剂	体积
前一步骤所得DNA样品	60 µl
连接缓冲液（LNB）	25 µl
NEBNext快速T4 DNA连接酶	10 µl
连接接头（LA）	5 µl
总体积	100 µl

弃掉磁珠。

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

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.

步骤结束

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

测序缓冲液（SB）
文库颗粒（LIB）
文库溶液（LIS）
测序芯片系绳（FCT）
测序芯片冲洗液（FCF）

耗材

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

仪器

PromethION测序设备
PromethION 测序芯片遮光片
P1000 移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头

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.

使用文库溶液

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

将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。

将测序芯片与连接器横竖对齐，以便顺利卡入。
用力下压芯片至卡槽，并确认卡夹位置归位。

重要

如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。

重要

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

准备就绪后，合上PromethION设备上盖。

请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。

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)
测序缓冲液（SB）
文库颗粒（LIB）
文库溶液（LIS）
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

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)
测序芯片冲洗液（FCF）
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

耗材

1.5 ml Eppendorf DNA LoBind离心管

仪器

P1000移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头

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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

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)
测序缓冲液（SB）
文库溶液（LIS）
文库颗粒（LIB）
Flow Cell Tether (FCT)
Flow Cell Flush (FCF)

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

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)
测序芯片冲洗液（FCF）
Loading Solution UL (LSU)
Sequencing Buffer UL (SBU)

耗材

1.5 ml Eppendorf DNA LoBind离心管

仪器

P1000移液枪和枪头
P200 移液枪和枪头
P20 移液枪和枪头

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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

步骤结束

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)
测序缓冲液（SB）
文库溶液（LIS）
文库颗粒（LIB）
Flow Cell Flush (FCF)
Flow Cell Tether (FCT)

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

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)
测序缓冲液（SB）
文库颗粒（LIB）
文库溶液（LIS）
测序芯片系绳（FCT）
测序芯片冲洗液（FCF）

耗材

1.5 ml Eppendorf DNA LoBind 离心管

仪器

P1000 移液枪和枪头
P20 移液枪和枪头
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.

提示

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

LIB管内的文库颗粒分散于悬浮液中。由于颗粒沉降速度非常快，因此请在混匀颗粒后立即使用。

对于大多数测序实验，我们建议您使用文库颗粒（LIB）。但如文库较为粘稠，您可考虑使用文库溶液（LIS）。

试剂	每张测序芯片的上样体积
测序缓冲液 (SB)	100 µl
文库颗粒 (LIB)，使用前充分混匀；或文库溶液 (LIS)	68 µl
DNA 文库	32 µl
总体积	200 µl

请注意: 此处增大了文库的上样量，以增强纳米孔阵列的覆盖度。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

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

从测序芯片中反旋排出缓冲液。请勿吸出超过20-30µl的缓冲液，并确保芯片上的纳米孔阵列一直有缓冲液覆盖。将气泡引入阵列会对纳米孔造成不可逆转地损害。

将P1000移液枪转至200µl刻度。
将枪头垂直插入加液孔中。
反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。

重要

为获得最佳测序产出，在文库样本上样后，请立即在测序芯片上安装遮光片。

我们建议在清洗芯片并重新上样时，将遮光片保留在测序芯片上。一旦文库从测序芯片中吸出，即可取下遮光片。

将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。

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.

步骤结束

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. 测序芯片的重复利用及回收

材料

测序芯片清洗剂盒（EXP-WSH004）

您可在纳米孔社区获取测序芯片清洗试剂盒实验指南。

提示

我们建议您在停止测序实验后尽快清洗测序芯片。如若无法实现，请将芯片留在测序设备上，于下一日清洗。

您可在此处找到回收测序芯片的说明。

重要

如果您遇到问题或对测序实验有疑问，请参阅本实验指南在线版本中的“疑难解答指南”一节。

31. DNA/RNA提取和文库制备过程中可能出现的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

低质量样本

现象	可能原因	措施及备注
低纯度DNA（Nanodrop测定的DNA吸光度比值260/280<1.8，260/230 <2.0-2.2）	用户所使用的DNA提取方法未能达到所需纯度	您可在污染物专题技术文档中查看污染物对后续文库制备和测序实验的影响。请尝试其它不会导致污染物残留的提取方法。请考虑将样品再次用磁珠纯化。
RNA完整度低（RNA完整值（RIN）<9.5，或rRNA在电泳凝胶上的条带呈弥散状）	RNA在提取过程中降解	请尝试其它 RNA 提取方法。您可在 RNA完整值专题技术文档中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅 DNA/RNA 操作页面。
RNA的片段长度短于预期	RNA在提取过程中降解	请尝试其它 RNA 提取方法。您可在 RNA完整值专题技术文档中查看更多有关RNA完整值（RIN）的介绍。更多信息，请参阅DNA/RNA 操作页面。我们建议用户在无RNA酶污染的环境中操作，并确保实验设备没有受RNA酶污染.

经AMPure磁珠纯化后的DNA回收率低

现象	可能原因	措施及备注
低回收率	AMPure磁珠量与样品量的比例低于预期，导致DNA因未被捕获而丢失	1. AMPure磁珠的沉降速度很快。因此临加入磁珠至样品前，请确保将磁珠重悬充分混匀。 2. 当AMPure磁珠量与样品量的比值低于0.4:1时，所有的DNA片段都会在纯化过程中丢失。
低回收率	DNA片段短于预期	AMPure磁珠量与样品量的比值越低，针对短片段的筛选就越严格。每次实验时，请先使用琼脂糖凝胶（或其他凝胶电泳方法）确定起始DNA的长度，并据此计算出合适的AMPure磁珠用量。
末端修复后的DNA回收率低	清洗步骤所用乙醇的浓度低于70%	当乙醇浓度低于70%时，DNA会从磁珠上洗脱下来。请确保使用正确浓度的乙醇。

32. 测序过程中可能出现的问题

以下表格列出了常见问题，以及可能的原因和解决方法。

我们还在 Nanopore 社区的“Support”板块提供了常见问题解答（FAQ）。

如果以下方案仍无法解决您的问题，请通过电邮(support@nanoporetech.com)）或微信公众号在线支持（NanoporeSupport）联系我们。

Mux扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数

现象	可能原因	措施及备注
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数	纳米孔阵列中引入了气泡	在对通过质控的芯片进行预处理之前，请务必排出预处理孔附近的气泡。否则，气泡会进入纳米孔阵列对其造成不可逆转地损害。视频中演示了避免引入气泡的最佳操作方法。
MinKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数	测序芯片没有正确插入测序仪	停止测序，将芯片从测序仪中取出，再重新插入测序仪内。请确保测序芯片被牢固地嵌入测序仪中，且达到目标温度。如用户使用的是GridION/PromethION测序仪，也可尝试将芯片插入仪器的其它位置进行测序。
inKNOW Mux 扫描在测序起始时报告的活性孔数少于芯片质检时报告的活性孔数	文库中残留的污染物对纳米孔造成损害或堵塞	在测序芯片质检阶段，我们用芯片储存缓冲液中的质控DNA分子来评估活性纳米孔的数量。而在测序开始时，我们使用DNA文库本身来评估活性纳米孔的数量。因此，活性纳米孔的数量在这两次评估中会有约10%的浮动。如测序开始时报告的孔数明显降低，则可能是由于文库中的污染物对膜结构造成了损坏或将纳米孔堵塞。用户可能需要使用其它的DNA/RNA提取或纯化方法，以提高起始核酸的纯度。您可在污染物专题技术文档中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。

MinKNOW脚本失败

现象	可能原因	措施及备注
MinKNOW显示 "Script failed”（脚本失败）		重启计算机及MinKNOW。如问题仍未得到解决，请收集 MinKNOW 日志文件并联系我们的技术支持。如您没有其他可用的测序设备，我们建议您先将装有文库的测序芯片置于4°C 储存，并联系我们的技术支持团队获取进一步储存上的建议。

纳米孔利用率低于40%

现象	可能原因	措施及备注
纳米孔利用率<40%	测序芯片中的文库量不足	请确保您按照相应实验指南，向测序芯片中加入正确浓度和体积的测序文库。请在上样前对文库进行定量，并使用 Promega Biomath Calculator 等工具中的“ dsDNA：µg to pmol”功能来计算DNA分子的摩尔量。
纳米孔利用率接近0	使用连接测序试剂盒，但接头并未与DNA成功连接	请确保您在“测序接头连接”步骤中使用的是NEBNext快速连接模块（E6056），以及SQK-LSK114试剂盒中的连接缓冲液（LNB）。同时，请确保每种试剂的用量正确。您可通过制备Lambda对照文库来检验第三方试剂的可用性。
纳米孔利用率接近0	使用连接测序试剂盒；但在接头连接后的纯化步骤中并未使用LFB 或SFB洗涤，而是使用了酒精	酒精可导致测序接头上的马达蛋白变性。请确保在测序接头连接后使用LFB或SFB。
纳米孔利用率接近0	测序芯片中无系绳	系绳是随着预处理液加至芯片的（试剂盒9、10和11系列对应冲洗系绳FLT；试剂盒14系列对应测序芯片系绳FCT）。请确保您在制备预处理液时，按需将FLT或FCT加入冲洗缓冲液（对应试剂盒9、10和11系列）或测序芯片冲洗液（对应试剂盒14系列）中。

读长短于预期

现象	可能原因	措施及备注
读长短于预期	DNA样本降解	读长反映了起始DNA片段的长度。起始DNA在提取和文库制备过程中均有可能被打断。 1. 1. 请查阅纳米孔社区中的提取方法以获得最佳DNA提取方案。 2. 在进行文库制备之前，请先跑电泳，查看起始DNA片段的长度分布。在上图中，样本1为高分子量DNA，而样本2为降解样本。 3. 在制备文库的过程中，请避免使用吹打或/和涡旋振荡的方式来混合试剂。轻弹或上下颠倒离心管即可。

大量纳米孔处于不可用状态

现象	可能原因	Comments and actions
大量纳米孔处于不可用状态 (在通道面板和纳米孔活动状态图上以蓝色表示) 上方的纳米孔活动状态图显示：状态为不可用的纳米孔的比例随着测序进程而不断增加。	样本中含有污染物	使用MinKNOW中的“Unblocking”（疏通）功能，可对一些污染物进行清除。如疏通成功，纳米孔的状态会变为"测序孔". 若疏通后，状态为不可用的纳米孔的比例仍然很高甚至增加： 1. 用户可使用测序芯片冲洗试剂盒（EXP-WSH004）进行核酸酶冲洗 can be performed, 操作，或 2. 使用PCR扩增目标片段，以稀释可能导致问题的污染物。

大量纳米孔处于失活状态

现象	可能原因	措施及备注
大量纳米孔处于失活状态（在通道面板和纳米孔活动状态图上以浅蓝色表示。膜结构或纳米孔遭受不可逆转地损伤）	测序芯片中引入了气泡	在芯片预处理和文库上样过程中引入的气泡会对纳米孔带来不可逆转地损害。请观看测序芯片的预处理及上样视频了解最佳操作方法。
大量纳米孔处于失活/不可用状态	文库中存在与DNA共纯化的化合物	与植物基因组DNA相关的多糖通常能与DNA一同纯化出来。 1. 请参考植物叶片DNA提取方法。 2. 使用QIAGEN PowerClean Pro试剂盒进行纯化。 3. 利用QIAGEN REPLI-g试剂盒对原始gDNA样本进行全基因组扩增。
大量纳米孔处于失活/不可用状态	样本中含有污染物	您可在污染物专题技术文档中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。

温度波动

现象	可能原因	措施及备注
温度波动	测序芯片和仪器接触不良	检查芯片背面的金属板是否有热垫覆盖。重新插入测序芯片，用力向下按压，以确保芯片的连接器引脚与测序仪牢固接触。如问题仍未得到解决，请联系我们的技术支持。

未能达到目标温度

现象	可能原因	措施及备注
MinKNOW显示“未能达到目标温度”	测序仪所处环境低于标准室温，或通风不良（以致芯片过热）	MinKNOW会限定测序芯片达到目标温度的时间。当超过限定时间后，系统会显示出错信息，但测序实验仍会继续。值得注意的是，在错误温度下测序可能会导致通量和数据质量（Q值）降低。请调整测序仪的摆放位置，确保其置于室温下、通风良好的环境中后，再在MinKNOW中继续实验。有关MinION温度控制的更多信息，请参考此 FAQ （常见问题）文档。

Device:

London Calling 2025

所有产品

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

疑难解答指南

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

疑难解答指南

概览

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

耗材

仪器

可选仪器

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.

第三方试剂

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. 计算机要求及软件

PromethION 24/48 的IT配置要求

PromethION 2 Solo 的IT配置要求

纳米孔测序相关软件

MinKNOW

EPI2ME （可选）

测序芯片质检

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

耗材

仪器

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.

步骤结束

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

耗材

仪器