Telomere-to-telomere sequencing (T2T) on PromethION (SQK-APK114, SQK-LSK114, and SQK-ULK114)

概览

For Research Use Only

This is a registration-based Early Access product.

Document version: T2T_9211_v114_revE_04Sep2024

1. Overview of the protocol

重要

This is a registration-based Early Access product

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.

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

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, using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114), the Assembly Polishing Kit V14 (SQK-APK114), and the Pore-C protocol with the Ligation Sequencing Kit V14 (SQK-LSK114). A total of four PromethION Flow Cells are recommended for telomere-to-telomere sequencing of a single human sample.

The protocol describes each experiment individually; however, the Ultra-Long DNA Sequencing Kit and Pore-C library preparation steps will be carried out across multiple days. All optional and required pause steps will be highlighted throughout the protocol.

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

To achieve in-depth telomere-to-telomere sequencing of a sample, three different datasets must be generated to give high-accuracy data, ultra-long reads, as well as chromatin conformation capture data.

The following three experiments are set up:

  • Ultra-long DNA sequencing experiment: This experiment yields ultra-long reads using the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114), generating a viscous library of ultra-long DNA fragments that requires careful handling to maintain the long fragments. The DNA extraction and library preparation processes are performed in duplicate side-by-side, taking ~3.5 hours with an overnight elution, yielding a total of ten library loads across two flow cells. This means five library loads are required per flow cell across a 140-hour sequencing run.

  • Assembly Polishing Kit experiment: This experiment generates high-accuracy data using the Assembly Polishing Kit V14 (SQK-APK114). It takes approximately one day to complete the library preparation step and sequencing is performed on one PromethION Flow Cell.

  • Pore-C sequencing experiment: This experiment generates chromatin conformation capture data using the Pore-C protocol and the Ligation Sequencing Kit V14 (SQK-LSK114). The Pore-C DNA extraction takes ~3 hours of hands-on time over three days with two overnight steps. The library preparation step takes ~60 minutes of hands-on time and is loaded on one PromethION Flow Cell. 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 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:

  • Ensure you have your human cell line or whole blood samples ready.
  • Ensure you have your sequencing kits, the correct equipment, and third-party reagents.
  • Download the MinKNOW software for acquiring and analysing your data.
  • Check your flow cells to ensure they have enough pores for a good sequencing run.

Protocol workflow

The tables below are an overview of the steps required in each experiment, including timings and optional stopping points.

Ultra-long DNA experiment
Note: this experiment is performed in duplicate, each prep yielding five library loads per flow cell.

Steps Process Time Stop option
Cell isolation Isolate white blood cells from whole blood or cells from cell culture 30 minutes 4°C overnight
Ultra-long DNA extraction Extract ultra-long high molecular weight DNA 190 minutes Stored at room temperature overnight, store at 4°C for short-term storage
Library preparation Tagment your DNA using a diluted fragmentation mix, attach the sequencing adapters and clean up the sample be precipitating your DNA and eluting overnight 190 minutes Overnight elution at room temperature.

4°C short-term storage or for repeated use, such as reloading your flow cell.

-80°C for single-use, long-term storage.

We strongly recommend sequencing your library as soon as it is adapted.

All excess adapted DNA library should be stored at 4°C or on ice until use.
Priming and loading your flow cell Prime your flow cell and load the prepared library for sequencing ~ 30 minutes
(5 minutes hands-on time)
Washing and reloading your flow cell Wash your flow cell and reload the prepared library for further sequencing every 20-24 hours until you have sequenced five library loads on a flow cell. ~ 45 minutes
(5-10 minutes hands-on time)

Assembly Polishing Kit experiment

Steps Process Time Stop option
DNA extraction and shearing Extract DNA from either whole blood cells or cell culture. Shear the extracted DNA using the Covaris g-TUBE™ 225–300 minutes (hands-on time) For either extraction option you can store the DNA at 4°C until the shearing step.

Following DNA shearing, the sample can be stored at 4°C until library preparation.
Library preparation Repair DNA and prepare the DNA ends for adapter attachment for the single cycle polymerase fill-in step. Finally, attach the sequencing adapters for the DNA ends. 175 minutes (hands-on time) There are multiple optional pause steps at 4°C overnight.

We strongly recommend sequencing your library as soon as the Rapid Adapter (RA) is attached.
Priming and loading your flow cell Prime the flow cell and load the prepared library for sequencing. ~ 30 minutes
(5 minutes hands-on time)

Pore-C experiment

Steps Process Time Stop option
Cell isolation Isolate white blood cells from whole blood or cells from cell culture 160 minutes The custom SPRI beads can be made and stored at 4°C before use.

Snap Freeze aliquots of white blood cells and store at -80°C until the experiment can begin.
Pore-C extraction Crosslink the three-dimensional DNA interactions within the nucleus of isolated cells. Next, permeabilise the cells and denature the chromatin. Cleave the genome with a restriction enzyme and 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 and finally, purify the Pore-C extract. Day 1: 50 minutes hands on time 2.5 hour procedure time and overnight

Day 2: 10 minutes hands on time, 6 hours procedure time and overnight

Day 3: 40 minute hands on time, 1 hour 50 procedure time and optional overnight step
Aside from the multiple overnight incubations, there are a couple of optional pause steps.

Snap-freeze the crosslinked aliquots in liquid nitrogen.
Store frozen sample pellets at –80°C and use within one year.

The extracted DNA can be stored at 4°C overnight until library preparation.
Library preparation Repair DNA and prepare the ends for sequencing adapter attachment. 60 minutes Overnight storage at 4°C following DNA library elution.

We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading your flow cell Prime the flow cell and load the prepared library for sequencing. ~ 30 minutes
(5 minutes hands-on time)

Sequencing and analysis

  • For each experiment, start a sequencing run using the MinKNOW software which will collect raw data from the device. Live basecalling is performed to support output estimates during sequencing.

Further details for each experiment set-up and basecalling are outlined in the "Data acquisition and basecalling" section of each experiment:

  • For the Ultra-long DNA experiment re-basecall your data after sequencing has completed using Dorado.
  • For the Assembly Polishing Kit experiment re-basecall your data after sequencing has completed using Dorado, then perform read correction of your basecalled data using the dorado correct command.
  • For the Pore-C experiment proceed with the data that has been is basecalled live in MinKNOW during sequencing.

Telomere-to-telomere assembly:

  • Finally, perform the telomere-to-telomere assembly using your basecalled data from all three experiments following the "Downstream analysis" section of the protocol.
重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Assembly Polishing Kit V14 (SQK-APK114)
  • Ligation Sequencing Kit V14 (SQK-LSK114)
  • Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
  • Ultra-Long Auxiliary Vials (EXP-ULA001)
  • Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL)
  • Flow Cell Priming Kit V14 (EXP-FLP004)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)
  • R10.4.1 PromethION Flow Cells (FLO-PRO114M)
  • PromethION 24/48 device - PromethION IT requirements document

2. Equipment and consumables

材料
  • 10–15 ml of whole blood
  • Assembly Polishing Kit V14 (SQK-APK114)
  • Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
  • 连接测序试剂盒V14(SQK-LSK114)
  • 测序芯片清洗剂盒(EXP-WSH004)或测序芯片清洗试剂盒 XL(EXP-WSH004-XL)
  • Ultra-Long Auxiliary Vials (EXP-ULA001)
  • Flow Cell Priming Kit (EXP-FLP002)
  • Sequencing Auxiliary Vials V14 (EXP-AUX003)

耗材
  • PromethION 测序芯片
  • 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)
  • 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)
  • NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
  • 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)
  • (Optional) chilled 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)
  • Salt-T4® DNA Ligase (NEB, M0467)
  • 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)
  • 1 M Tris-HCl, pH 7.5
  • Proteinase K at 20 μg/μl (NEB, P8107S)
  • 10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • Recombinant Albumin at 20 μg/μl (NEB, B9200S)
  • LongAmp® Hot Start Taq DNA Polymerase (NEB, M0534S/L)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Exonuclease I (NEB, M0293S/L)
  • 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)
  • 新制备的70%乙醇(用无核酸酶水配制)
  • 新制备的80%乙醇(用无核酸酶水配制)
  • 3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
  • 50 ml centrifuge tubes
  • 15 ml Falcon tubes
  • 5 ml centrifuge tubes
  • 2 ml Eppendorf DNA LoBind 离心管
  • 1.5 ml Eppendorf DNA LoBind离心管
  • 0.2 ml thin-walled PCR tubes
  • Qubit™ 分析管(Invitrogen, Q32856)
  • g-TUBE™ (Covaris, 520079)
  • Ziplock bags
  • 0.2 µm filter

仪器
  • PromethION 测序芯片遮光片
  • 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
  • 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
  • Eppendorf 5424 离心机(或等效器材)
  • Inoculation loop or disposable tweezers for spooling DNA
  • -80°C freezer storage
可选仪器
  • Liquid nitrogen and canister

We recommend performing this experiment with freshly extracted DNA from either human cell lines or fresh whole blood.

We recommend different sample preparations due to different input requirements for each experiment, with the option to use either human cell lines or human whole blood. Other methods are available and may be more appropriate for your lab; however, please ensure to yield enough input required for each library preparation. It is also worth noting that 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.

Human whole blood: approximately a total of 10–15 ml of blood is required for all sample preparation steps. 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.

Human whole blood input requirements:

  • Ultra-long DNA experiment: 3.2 ml
  • Assembly Polishing Kit experiment: 1 ml
  • Pore-C experiment: 5-10 ml

Human cell lines from culture can also be used. We recommend isolating them from cell culture using standard techniques. However, for the Assembly Polishing Kit experiment, we have included an extraction protocol from cell culture.

  • Ultra-long DNA experiment: 6 million cells
  • Assembly Polishing Kit experiment: 5 million cells
  • Pore-C experiment: 10 million cells

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.

注意

This protocol includes the use of potentially hazardous reagents. Please adhere to the correct health and safety practices in accordance to the manufacturers instructions and your laboratory standards.

Assembly Polishing Kit V14 (SQK-APK114) content

SQK-APK114

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
AP Adapter APA Blue 1 15
AP Primer APP Yellow 1 35
Rapid Adapter RA Green 1 15
Long Fragment Buffer LFB Clear 1 7,500
Ligation Buffer LNB White 1 200
AMPure XP Beads AXP Amber 3 1,200
Adapter Buffer ADB Clear 1 100
Elution Buffer EB Black 1 500
Sequencing Buffer SB Red 1 700
Library Beads LIB Pink 1 600
Library Solution LIS White cap, pink label 1 600
Flow Cell Flush FCF Clear cap, light blue label 1 8,000
Flow Cell Tether UL FTU Purple 1 600
AP Mix APM Clear cap 1 60

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

ULK114 tubes

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

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

EXP-ULA001 Kit content

Name Acronym Cap colour Number of vials Fill volume per vial (µl)
Elution Buffer EB Black 1 1,500
Sequencing Buffer UL SBU Red 2 1,000
Loading Solution UL LSU White cap, pink label 1 200
Flush Tether UL FTU Purple 1 600
Flow Cell Flush FCF Clear cap, light blue label 1 15,500

连接测序试剂盒V14(SQK-LSK114)内容物

请注意: 我们正在将部分试剂的包装形式由单次管装改为瓶装。

单次管装试剂: SQK-LSK114 v2

部分试剂改为瓶装: SQK-LSK114 v3

声明: 本产品包含由贝克曼库尔特公司(Beckman Coulter, Inc)生产的 AMPure XP 试剂,并可与试剂盒一起于-20°C 下储存(试剂稳定性将不受损害)。

请注意: DNA参照(DCS)是一段可比对到Lambda基因组的3'端、长度为3.6 kb 的标准扩增子。

Flow Cell Wash Kit (EXP-WSH004) contents

EXP-WSH004 kit contents v2

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

Flow Cell Priming Kit (EXP-FLP004) contents

EXP-FLP004v2

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Flow Cell Flush FCF 6 Clear cap, light blue lable 8,000
Flow Cell Tether FCT 1 Purple 200

Sequencing Auxiliary Vials V14 (EXP-AUX003) contents

EXP-AUX003 bottles

Name Acronym Cap colour No. of vials Fill volume per vial (μl)
Elution Buffer EB Black 2 500
Sequencing Buffer SB Red 2 700
Library Solution LIS White cap, pink label 2 600
Library Beads LIB Pink 2 600
Flow Cell Flush FCF Light blue label 2 8,000
Flow Cell Tether FCT Purple 2 200

3. Sample preparation: whole blood cell isolation

材料
  • 1.6 ml of whole blood (x2, one for each duplicate preparation)

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
  • 无核酸酶水(如ThermoFisher,AM9937)

仪器
  • 迷你离心机
  • P1000 移液枪和枪头
  • P200 移液枪和枪头
  • P20 pipette and tips
  • Eppendorf 5424 离心机(或等效器材)

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

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

Users may isolate white blood cells by any means they think are most appropriate for the whole blood sample to be used. If an alternative method is used, this step can be skipped and proceed directly to the next section of the protocol.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate and we recommend performing this step for both volumes of blood side by side. Note: ensure both blood volumes are from the same sample.

In a fresh 15 ml Falcon tube, prepare 10 ml of 1x PBS in nuclease-free water as follows:

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

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

Gently invert the tube ten times to mix.

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

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

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

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

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

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

提示

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

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

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

步骤结束

Take forward 6 million white blood cells forward into the next step. Store the pellet at 4°C until the experiment can begin.

4. Sample preparation: ultra-long DNA extraction

材料
  • 6 million white blood cells isolated from whole blood (x2, one for each duplicate preparation)
  • 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
  • Thermomixer
  • 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

Ultra-long DNA extraction

In this step, ultra-long DNA is extracted from the isolated cells and quantified before going into the library preparation step.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate and we recommend performing this step for both white blood cells isolated from whole blood side by side.

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

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

重要

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

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

Transfer 6 million cells resuspended in 40 µl PBS to a fresh 5 ml tube.

提示

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

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

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

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

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

Incubate the reaction at 56°C for 10 minutes.

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

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

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

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

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

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

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

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

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.

Add three Monarch DNA Capture Beads to the collected DNA phase.

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

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

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.

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

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

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

Prepare the Monarch gDNA Wash Buffer with ethanol.

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

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

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

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

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

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

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

Insert a Monarch Bead Retainer into a Monarch Collection Tube II and transfer the beads into the retainer.

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

重要

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

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

重要

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

Incubate the tube for 10 minutes at 56°C and insert a fresh Monarch Bead Retainer into a fresh Monarch Collection Tube II.

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

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

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

Incubate the eluate for 10 minutes at 56°C.

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

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

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.

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

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.

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

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

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

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

CHECKPOINT

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

步骤结束

Take forwards 750 µl of extracted ultra-long DNA into the library preparation step. Store the DNA on ice until the next step can begin.

5. Library preparation: ultra-long DNA sequencing

材料
  • 750 µl of extracted uHMW gDNA in EEB (x2, one for each duplicate preparation)
  • Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)

耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管

仪器
  • Thermal cycler or heat block
  • 迷你离心机
  • Vortex mixer
  • Hula混匀仪(低速旋转式混匀仪)
  • Eppendorf 5424 离心机(或等效器材)
  • 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

Library preparation for the ultra-long DNA experiment

In this step, the extracted ultra-long DNA is prepared for sequencing by tagmentation and rapid attachment of sequencing adapters.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates of 750 µl of extracted uHMW gDNA in EEB.

Thaw, spin down and pipette mix the Fragmentation Mix (FRA), FRA Dilution Buffer (FDB), and Rapid Adapter (RA) and store on ice.

Pre-heat a thermal cycler or heat block to 75ºC.

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

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

Mix the diluted Fragmentation Mix (FRA) by pipetting.

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

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

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

Incubate the reaction as follows:

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

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

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

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

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

Incubate the reaction for 30 minutes at room temperature.

Thaw the Precipitation Buffer (PTB) and Elution Buffer (EB) at room temperature before spinning down and pipette mixing the reagents. Once thawed, store the reagents on ice.

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

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

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

Centrifuge the sample at 1000 x g for 1 minute.

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

Centrifuge the sample at 1000 x g for 1 minute.

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

Using a regular pipette tip, add 480 µl of Elution Buffer (EB) to the tube containing the DNA. Incubate overnight at room temperature, for a minimum of 12 hours.

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

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

步骤结束

After overnight incubation, the DNA library can be taken forwards into flow cell priming and loading. Store the library at 4°C for short-term storage as the flow cell will need to be loaded with the same library five times.

6. 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 测序芯片
  • 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

Priming and loading the flow cell to sequencing ultra-long DNA

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.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates.

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。

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

In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the DNA library for loading as follows using a wide-bore pipette tip for the addition of the DNA library:

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.

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

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

Prepare the flow cell priming mix in a 1.5 ml Eppendorf DNA LoBind tube and mix by vortexing at room temperature.

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

对PromethION 24/48,将测序芯片插入相应卡槽的对接端口:

  1. 将测序芯片与连接器横竖对齐,以便顺利卡入。

  2. 用力下压芯片至卡槽,并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

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。

Screenshot 2021-04-08 at 12.08.37

顺时针滑动加液孔孔盖,将其打开。

Prom Flowcell Loading 2 中文

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。

在加液孔打开的状态下,按下述步骤吸取少量液体,同时避免引入气泡:

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  3. 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。

Prom Flowcell Loading 3 中文

Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes.

Step 4 v1

缓慢向芯片的加液口中加入500 µl预处理液,完成芯片的预处理。

Prom Flowcell Loading 5 中文

Ensure the inlet port cover of the flow cell is still open in preparation for loading.

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).

PromethION Step 1

重要

Take care when loading the flow cell as the DNA library is very viscous and may not readily flow through the inlet port, requiring extra careful pipetting to prevent introducing air bubbles.

Aspirate the DNA library with a wide-bore pipette tip and ensure there are no air bubbles in the tip. Place the wide-bore pipette tip directly on the inlet port. Slowly depress the pipette to dispense the library into the inlet port.

The DNA library is viscous and 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 leave 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. Due to the viscosity of the DNA library, a drop may sit on the inlet port.

If the DNA library is not fully absorbed into the inlet port, use a P200 pipette, set it to 50 µl and insert the tip into port 2.

Very slowly turn the wheel of the pipette to create a negative pressure in the flow cell. This will pull the DNA library into the inlet port. Closely watch the DNA library and completely remove the pipette as soon as the library starts to be pulled into the port.

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

Close the valve to seal the inlet port.

Step 7 V2

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。

如遮光片不在测序芯片上,请您按照以下步骤安装:

  1. 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。
  2. 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。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

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。

7. Washing and reloading the PromethION Flow Cell with ultra-long DNA library

材料
  • Flow Cell Wash Kit (EXP-WSH004) or 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

Flow cell washing and reloading for the ultra-long DNA experiment

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 five 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.

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.

image (109)

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.

As explained in the introduction, the ultra-long DNA experiment must be performed in duplicate. This step can be performed side by side or separately for both duplicates.

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.

Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.

Thaw one tube of Wash Diluent (DIL) at room temperature.

Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, then spin down briefly and place on ice.

In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the following Flow Cell Wash Mix:

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

Mix well by pipetting, and place on ice. Do not vortex the tube.

Pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.

Ensure the inlet port is closed and remove the buffer from the waste port, using a P1000 pipette.

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

Picture7

Slide the inlet port cover clockwise to open the inlet port.

Step 2 V2

After opening the inlet port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:

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

Step 3 v1

Slowly load 200 µl of the prepared flow cell wash mix into the inlet port, as follows:

  1. Using a P1000 pipette, take 200 µl of the flow cell wash mix
  2. Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
  3. 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.
  4. Set a timer for a 5 minute incubation.

Once the 5 minute incubation time is complete, carefully load the remaining 200 µl of the prepared flow cell wash mix into the inlet port, as follows:

  1. Using a P1000 pipette, take 200 µl of the flow cell wash mix
  2. Insert the pipette tip into the inlet port, ensuring there are no bubbles in the tip
  3. 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.

Close the inlet port and wait for 1 hour.

Step 7 V2 edited to step 5

Ensure the inlet port is closed and remove buffer from the waste port a second time.

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

Picture7

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.

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

In a fresh 1.5 ml Eppendorf DNA LoBind tube, prepare the DNA library for loading as follows using a wide-bore pipette tip for the addition of the DNA library:

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.

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

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

Prepare the flow cell priming mix in a 1.5 ml Eppendorf DNA LoBind tube and mix by vortexing at room temperature.

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

Slide the inlet port cover clockwise to open.

Prom loading 2

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。

在加液孔打开的状态下,按下述步骤吸取少量液体,同时避免引入气泡:

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  3. 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。

Prom Flowcell Loading 3 中文

重要

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

Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes.

Step 4 v1

Turn the valve to close the inlet port and use a P1000 to remove all fluid from the waste channel through one of the waste ports.

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

Slide open the inlet port and load 500 µl of the priming mix into the flow cell via the inlet port to complete a second flow cell flush, avoiding the introduction of air bubbles.

Step 5 v1

Close the inlet port and use a P1000 to remove all fluid from the waste channel through a waste port again.

Open the inlet port cover of the flow cell in preparation for loading.

PromethION Step 1

重要

Take care when loading the flow cell as the DNA library is very viscous and may not readily flow through the inlet port, requiring extra careful pipetting to prevent introducing air bubbles.

Aspirate the DNA library with a wide-bore pipette tip and ensure there are no air bubbles in the tip. Place the wide-bore pipette tip directly on the inlet port. Slowly depress the pipette to dispense the library into the inlet port.

The DNA library is viscous and 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 leave 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. Due to the viscosity of the DNA library, a drop may sit on the inlet port.

If the DNA library is not fully absorbed into the inlet port, use a P200 pipette, set it to 50 µl and insert the tip into port 2.

Very slowly turn the wheel of the pipette to create a negative pressure in the flow cell. This will pull the DNA library into the inlet port. Closely watch the DNA library and completely remove the pipette as soon as the library starts to be pulled into the port.

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

Close the valve to seal the inlet port.

Step 7 V2

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。

如遮光片不在测序芯片上,请您按照以下步骤安装:

  1. 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。
  2. 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。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

步骤结束

Resume the sequencing run on MinKNOW to continue data acquisition.

8. Data acquisition and basecalling: ultra-long DNA

重要

Ensure you are using the most recent version of MinKNOW.

We recommend updating MinKNOW to the latest version prior to starting a sequencing run for the best sequencing results.

For more information on updating MinKNOW, please refer to our MinKNOW protocol.

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

We recommend first basecalling in real-time using the fast basecaller on MinKNOW using the PromethION 24 or 48 device. MinKNOW can be used and set up to sequence in multiple ways:

  • On a computer either directly or remotely connected to a sequencing device.
  • Directly on a PromethION 24/48 sequencing device.

After real-time basecalling, rebasecall the data using the super-accurate (SUP) basecaller v5.0 model (or newer) in Dorado.

For more information on using MinKNOW on a sequencing device, please see the PromethION 24/48 user manual.


Real-time sequencing

To start a run on MinKNOW to sequence ultra-long DNA

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) on the Kit page.

4.

Configure the sequencing parameters as follows:
Basecalling: off
Modified bases: off
Model: fast basecalling
Barcoding: off
Alignment: off
Adaptive sampling: off
Advanced options: default settings

5.

Configure the data targets as follows:
Run duration: 140 hours

6.

Configure the analysis workflow:
Workflow: off

7.

Configure the output parameters as follows:
Basecalled output type: .BAM
Based on: Time elapsed
Frequency: Every 10 minutes
FASTQ options - Compression: on
Raw reads: on
POD5: on
FAST5: off

8.

Configure the filterning options as follows:
Filtering: on
Min Qscore: 10
Min read length (kb): 1

Post-sequencing basecalling

Once sequencing is complete, rebasecall your data using the super-accurate (SUP) basecaller using command line Dorado with the following commands as described on the Dorado Github page:

$ dorado basecaller --min-qscore 10 sup ultralong_pod5s/ > ulk_reads.bam

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

Please remember to change the output file name to differentiate between each of the ultra-long experiment flow cells run in duplicate.

In the Downstream analysis section, we outline further options for analysing your basecalled data for the telomere-to-telomore experiment.

9. Sample preparation: human cell line DNA extraction (Option 1)

材料
  • 5 million cells (e.g. cell culture or tissue sample)

耗材
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Puregene Cell Kit (QIAGEN, 158043)
  • 新制备的70%乙醇(用无核酸酶水配制)
  • Isopropanol, 100% (Fisher, 10723124)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • 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)

仪器
  • Eppendorf 5424 离心机(或等效器材)
  • 迷你离心机
  • Thermal cycler or heat block
  • Thermomixer
  • 涡旋混匀仪
  • Inoculation loop or disposable tweezers for spooling DNA
  • Wide-bore pipette tips
  • Qubit荧光计(或用于质控检测的等效仪器)
  • Hula混匀仪(低速旋转式混匀仪)
  • 盛有冰的冰桶
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P100移液枪和枪头
  • P20 移液枪和枪头

DNA extraction from human cell lines for the Assembly Polishing Kit (SQK-APK114) experiment

An input of 5 μg of gDNA must be prepared for the Assembly Polishing Kit experiment. Below we outline how to use the QIAGEN Puregene Cell Kit to extract your input.

Prepare a 1.5 ml Eppendorf DNA LoBind tube with 1 ml of 70% ethanol and store on ice to cool.

Harvest and pellet 5 million cells in a 1.5 ml Eppendorf DNA LoBind tube. If any liquid remains associated with the pellet, spin down again, then aspirate and discard the remaining supernatant.

Add 200 µl of 1x phosphate buffered saline (PBS) to the pelleted cells.

Aspirate and discard the supernatant without disturbing the pellet.

Add 2 ml of Cell Lysis Solution to the cell pellet and resuspend using a wide-bore pipette tip.

Transfer the resuspended cells to a 15 ml Falcon tube. If any cell clumps remain, gently invert the tube until the cells are fully resuspended.

Incubate the sample at 37°C for 30 minutes.

Add 700 µl Protein Precipitation Solution to the lysed cells and mix by vortexing with three pulses of 5 seconds.

Centrifuge at 2000 x g for 5 minutes.

Carefully transfer the supernatant to a fresh 15 ml Falcon tube and discard the pellet.

Add 2.5 ml of room temperature isopropanol and mix by gently inverting the tube ~50 times.

Prepare 250 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) in a fresh 1.5 ml Eppendorf DNA LoBind tube.

Spool the DNA using an inoculation loop or disposable tweezers.

Briefly dip the spooled DNA into the cold 70% ethanol and allow the DNA to air dry for a few seconds.

Transfer the spooled DNA to the previously prepared TE buffer and allow the DNA to gently dislodge from the inoculation loop or tweezers.

Incubate the DNA pellet for 2 hours at 50°C, occasionally mixing the tube by gentle inversion to aid dissolving the pellet. Alternatively, the DNA pellet can be left overnight at room temperature on a Hula mixer.

Note: it is vital that the DNA pellet is completely dissolved and that the sample is homogenous before the quantification step.

CHECKPOINT

Quantify the sample three times using the Qubit dsDNA BR Assay Kit, ensuring that the replicate measurements are consistent before continuing to the next step.

Note: If your Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended.

步骤结束

Take forward 5 µg of extracted high molecular weight DNA into the shearing step. Store the DNA at 4°C until the next step can begin.

10. Sample preparation: whole blood DNA extraction (Option 2)

材料
  • 1 ml of whole blood

耗材
  • Puregene Blood Kit (QIAGEN, 158023)
  • Absorbent material e.g. paper towel or tissues
  • Freshly prepared 70% ethanol in nuclease-free water
  • Isopropanol, 100% (Fisher, 10723124)
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
  • 无核酸酶水(如ThermoFisher,AM9937)
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit™ 分析管(Invitrogen, Q32856)
  • 15 ml Falcon tubes
  • 1.5 ml Eppendorf DNA LoBind离心管

仪器
  • Centrifuge and rotor suitable for 15 ml Falcon tubes
  • Incubator or water bath set at 37°C and 50°C
  • 涡旋混匀仪
  • 迷你离心机
  • Qubit荧光计(或用于质控检测的等效仪器)
  • 盛有冰的冰桶
  • 计时器
  • Wide-bore pipette tips
  • P1000移液枪和枪头
  • P200 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
  • P2 移液枪和枪头
可选仪器
  • Agilent Femto Pulse System (or equivalent for read length QC)

DNA extraction from whole blood for the Assembly Polishing Kit (SQK-APK114) experiment

An input of 5 µg of gDNA must be prepared for the Assembly Polishing Kit experiment. Below we outline how to use the QIAGEN Puregene Cell Kit to extract your input.

Add 1 ml of whole blood to a clean 15 ml falcon tube.

Add 3 ml of RBC Lysis Solution into the 15ml falcon tube containing the blood.

Mix by inverting the tube 10 times.

Incubate for 5 minutes at room temperature (15–25°C). 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, ensuring your leave approximately 200 µl of the residual liquid and the white blood cell pellet.

Note: The supernatant can be removed by pipetting or by pouring the volume out on to an absorbent material.

Gently flick the tube and/or pipette mix using a wide bore tip to resuspend the pellet in the residual liquid.

Note: The pellet should be completely dispersed, this greatly facilitates the cell lysis in the next step.

Add 3 ml of Cell Lysis Solution.

Note: Thoroughly mix the reaction by pipette mixing after the addition of the Cell Lysis Solution.

Incubate the reaction at 37°C until no clumps remain.

Note: Ensure the solution is homogenous by the end of the incubation.
If necessary, you can mix the reaction by pipette mixing during the incubation to assist with homogenisation.

Add 15 μl of RNase A solution and incubate the reaction for 15 minutes at 37°C.

Transfer the reaction to ice bucket with ice, and incubate for 3 min to quickly cool the sample.

Add 1 ml of Protein Precipitation Solution to your sample. Invert the tube 10–20 times, until the solution is opaque.

Centrifuge your sample for 5 minutes at 2000 x g.

Note: The precipitated protein should form a tight, reddish-brown pellet. If the protein pellet is not tight, incubate the tube on ice for 5 minutes and repeat the centrifugation.

Pipette 3 ml of isopropanol into a clean 15 ml falcon tube.

Carefully pour the supernatant from the sample tube into the 15 ml falcon tube containing the isopropanol.

Note: Ensure that the protein pellet is not dislodged during pouring.

Gently mix the tube by inverting 50 times until the DNA is visible as threads or a clump.

Depending on how your DNA has aglomerated in the isopropanol, follow one set of the instructions below:

If the DNA is visible as a thread If the DNA is clumped
1. Spool the DNA using an inoculation loop and dip in ice-cold 70% ethanol.

2. Briefly allow to air dry.

3. Resuspend the spooled DNA in 200 µl TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).
1. Centrifuge at 2000 x g for 3 minutes, drain the tube and discard the supernatant by inverting on a clean piece of absorbent paper, ensuring the pellet remains.

2. Add 1 ml 70% ethanol and invert several times to wash the pellet.

3. Centrifuge for 1 minute at 2000 x g, discard the supernatant and drain the tube on a clean piece of absorbent paper, taking care that the pellet remains in the tube.

4. Air dry for 5–10 minutes.

5. Add 200 µl TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) to the pellet.

6. Vortex for 5 seconds at medium speed to resuspend the pellet.

Incubate the tube containing your DNA in 200 µl TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) for 1 hour at 65°C, occasionally mixing the tube contents by gentle inversion.

Note: The DNA pellet may take some time to solubilise, so ensure the solution is homogenous before quantifying.

Optional: Alternatively, this incubation can be performed at room temperature overnight.

Quantify your sample three times using the Qubit dsDNA BR Assay Kit. Ensure the replicate Qubit measurements are consistent before continuing to the next step.

步骤结束

Take forward 5 µg of extracted high molecular weight DNA into the shearing step. Store the DNA at 4°C until the next step can begin.

11. Sample preparation: shearing DNA for 10 kb input using the Covaris g-TUBE™

材料
  • 5 µg of extracted DNA

耗材
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Nuclease-free water
  • g-TUBE™ (Covaris, 520079)
  • Qubit™ 分析管(Invitrogen, Q32856)
  • 1.5 ml Eppendorf DNA LoBind离心管

仪器
  • Eppendorf 5424 离心机(或等效器材)
  • P200 移液枪和枪头
  • P20 移液枪和枪头
  • 盛有冰的冰桶

Shearing your DNA input for the Assembly Polishing Kit experiment

After DNA extraction from either cell culture or whole blood, we recommend shearing your DNA to 10 kb before library preparation.

Aliquot 5 µg DNA into an Eppendorf DNA LoBind 1.5 ml tube and make the total volume up to 150 µl with nuclease-free water.

Transfer the sample to a Covaris g-TUBE.

Centrifuge for 1 minute at 6000 RPM.

Note: if sample remains in the upper chamber, centrifuge again for 1 minute at 6000 RPM and repeat until all the sample passes into the lower chamber.

Turn the g-TUBE upside down and centrifuge for 1 minute at 6000 RPM.

Note: if sample remains in the upper chamber, centrifuge again for 60 seconds at 6000 RPM, and repeat until all the sample passes into the lower chamber.

Place the g-TUBE with the lid down into the stand and carefully unscrew the tube from the lid. Transfer the sheared sample to a 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT

Quantify the sample using the Qubit dsDNA BR Assay Kit to ensure that you have 5 µg of sheared DNA.

步骤结束

Take forward 5 µg of sheared DNA into the library preparation step. Store the DNA at 4°C until the next step can begin.

12. Library preparation: Assembly Polishing Kit

材料
  • 5 µg of sheared DNA
  • Assembly Polishing Kit V14 (SQK-APK114)

耗材
  • LongAmp® Hot Start Taq DNA Polymerase (NEB, M0534S/L)
  • NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
  • 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)
  • Salt-T4® DNA Ligase (NEB, M0467)
  • Exonuclease I (NEB, M0293S/L)
  • Qubit dsDNA HS Assay(双链DNA高灵敏度检测)试剂盒(Invitrogen, Q32851)
  • 新制备的80%乙醇(用无核酸酶水配制)
  • Nuclease-free water
  • 1.5 ml Eppendorf DNA LoBind离心管
  • 0.2 ml thin-walled PCR tubes

仪器
  • Hula混匀仪(低速旋转式混匀仪)
  • 迷你离心机
  • 磁力架
  • 涡旋混匀仪
  • Thermal cycler or heat block
  • Thermomixer
  • 盛有冰的冰桶
  • Qubit荧光计 (或用于质控检测的等效仪器)

Library preparation using the Assembly Polishing Kit

The library is prepared for sequencing using the Assembly Polishing Kit, where DNA nicks are repaired, and a single cycle polymerase step is performed to help achieve haplotype resolved Q50 assemblies. Finally, rapid adapters are attached to the DNA for sequencing.

根据生产厂家的说明准备NEBNext FFPE DNA 修复混合液和 NEBNext Ultra II 末端修复/ dA尾添加模块,并置于冰上。

为获得最优表现,NEB建议如下:

  1. 于冰上解冻所有试剂。

  2. 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。

  3. 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。

  4. 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。

  5. FFPE DNA 修复缓冲液可能轻微泛黄,不影响使用。

Thaw the AMPure XP Beads (AXP) at room temperature, briefly spin down and store on ice.

Prepare the DNA in nuclease-free water:

  1. Transfer 5 µg of sheared DNA into a 1.5 ml Eppendorf DNA LoBind tube.
  2. Adjust the volume to 240 µl with nuclease-free water.
  3. Mix thoroughly by pipette mixing or by flicking the tube.
  4. Briefly spin down the tube.

Prepare the following reaction in a 1.5 ml Eppendorf DNA LoBind tube.

Reagents Volume
Sheared gDNA 240 µl
NEBNext FFPE DNA Repair Buffer v2 35 µl
Ultra II End-prep Enzyme Mix 15 µl
NEBNext FFPE DNA Repair Mix 10 µl
Total 300 µl

Thoroughly mix the reaction by gently pipetting and briefly spin down the reaction.

Aliquot 100 µl of the reaction into three fresh 0.2 ml thin-walled PCR tubes and briefly spin down.

Incubate in a thermal cycler at 20°C for 5 minutes, then at 65°C for 5 minutes, and cool the reaction to 10°C.

Pool the aliquots into a fresh 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add 300 µl of resuspended AMPure XP Beads (AXP) to the reaction and incubate for 10 minutes on a Hula Mixer (rotator mixer) at room temperature.

Prepare at least 2 ml of fresh 80% ethanol in nuclease-free water.

Spin the reaction down and pellet the beads on a magnet until the supernatant is clear and colourless. Keep the tube on the magnet and pipette off the supernatant without disturbing the pellet.

Keep the tube on the magnet and wash the beads with 750 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove and discard the ethanol using a pipette.

Repeat the previous step.

Spin the tube down and place it back onto the magnet to pipette off any residual ethanol. Open the lid and allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnet and resuspend the pellet in 51 µl of nuclease-free water. Incubate at 37°C for 10 minutes and occasionally flick the tube to aid elution.

Pellet the beads on a magnet for at least 1 minute, until the eluate is clear and colourless.

Remove and retain the eluate into a fresh 0.2 ml thin-walled PCR tube.

可选操作

Quantify 1 µl of eluted sample using a Qubit fluorometer to QC.

可选操作

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

Thaw the AP Adapter (APA) at room temperature, briefly spin down and place on ice.

Spin down the Salt T4 DNA Ligase at room temperature, and place on ice.

Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to the viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.

Thaw the Long Fragment Buffer (LFB) at room temperature and mix by vortexing. Then spin down and place on ice.

In a 0.2 ml thin-walled PCR tube, prepare the following reaction:

Reagents Volume
End-prepped gDNA 50 µl
AP Adapter (APA) 2 µl
Salt T4 DNA Ligase 10 µl
Ligation Buffer (LNB) 25 µl
Nuclease-free water 13 µl
Total 100 µl

Thoroughly mix the reaction by gently pipetting and briefly spin down.

Incubate the reaction at 25°C for 30 minutes and at 65°C for 10 minutes, and then cool the reaction to 10°C.

Transfer the reaction to a new 1.5 ml Eppendorf DNA LoBind tube.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking.

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Spin the tube down and pellet on a magnet until the supernatant is clear and colourless. Keep the tube on the magnet and pipette off the supernatant.

Remove the tube from the magnet and wash the beads with 125 µl of Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, and then return the tube to the magnet and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Spin the tube down, place it back onto the magnet and pipette off any residual supernatant.

Remove the tube from the magnet and resuspend the pellet in 51 µl of nuclease-free water. Spin down the tube and incubate at 37°C for 10 minutes in a heat block, occasionally flicking the tube to aid elution.

Note: elution at 37°C can improve the recovery of long fragments from beads. Therefore we strongly recommend performing the elution at 37°C.

However, if a heat block or water bath is not available, this step can be performed at room temperature by increasing the incubation time to 15 minutes and flicking the tube to aid elution every 30 seconds.

Briefly spin down the tube and place it back onto the magnetic rack for at least 1 minute, until the eluate is clear and colourless.

Remove and retain 51 µl of eluate containing the DNA library into a fresh 0.2 ml thin-walled PCR tube.

Dispose of the pelleted beads.

可选操作

Quantify 1 µl of eluted sample using a Qubit fluorometer to QC.

可选操作

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

Thaw the AP Mix (APM) on ice, mix by vortexing, then spin down. Keep the tube on ice until use.

Thaw the AP Primer (APP), and 5x LongAmp Taq Buffer at room temperature and mix by vortexing. Then spin down and place on ice.

Spin down the LongAmp Taq HotStart Polymerase and ExoI, and place on ice.

In a fresh 0.2 ml thin-walled PCR tube, prepare the following reaction:

Reagents Volume
gDNA 50 µl
AP Mix (APM) 4 µl
AP Primer (APP) 5 µl
5x LongAmp Taq Buffer 20 µl
LongAmp Taq HotStart Polymerase 4 µl
Nuclease-free water 17 µl
Total 100 µl

Thoroughly mix the reaction by gently pipetting and briefly spin down.

Start the single cycle polymerase fill-in step by using the following conditions on a thermal cycler:

Temperature Time
94°C 3 minutes
57°C 5 minutes
65°C 40 minutes
10°C hold

Add 2 µl of ExoI (NEB, M0293) to the reaction and pipette mix thoroughly, then briefly spin down.

Incubate at 37°C for 15 minutes, and then at 80°C for 15 minutes on a thermal cycler.

Transfer the reaction to a clean 1.5 ml Eppendorf LoBind DNA tube.

Resuspend the AMPure XP Beads (AXP) by vortexting.

Add 40 µl of AMPure XP Beads (AXP) to the reaction.

Incubate on a Hula mixer (rotator mixer) for 10 minutes at room temperature.

Spin the reaction down and pellet on a magnet. Keep the tube on the magnet and pipette off the supernatant when it is clear and colourless.

Wash the beads by adding 125 µl Long Fragment Buffer (LFB). Flick the beads to resuspend and spin down the tube, then return it to the magnet. Allow the beads to pellet and remove and discard the supernatant with a pipette.

Repeat the previous step.

Spin the tube down, place it back onto the magnet and pipette off any residual supernatant.

Remove the tube from the magnet and resuspend the pellet in 51 µl of nuclease-free water. Spin down the reaction and incubate at 37°C for 10 minutes, occasionally flicking the tube to aid elution.

Pellet the beads on a magnet for at least one minute, until the eluate is clear and colourless.

Remove and retain 51 µl of eluate in a fresh 1.5 ml Eppendorf DNA LoBind tube.

Dispose of the pelleted beads.

可选操作

Quantify 1 µl of eluted sample using a Qubit fluorometer to QC.

可选操作

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

Thaw the Adapter Dilution Buffer (ADB) and Elution Buffer (EB) at room temperature, spin down, and mix by pipetting.

Spin down the Rapid Adapter (RA) and place on ice.

In a fresh 1.5 ml Eppendorf DNA LoBind tube, dilute the Rapid Adapter (RA) as follows:

Reagents Volume
Rapid Adapter (RA) 1.5 µl
Adapter Dilution Buffer (ADB) 3.5 µl
Total 5 µl

Mix the dilution by gently pipetting the full volume.

Add 5 µl of diluted Rapid Adapter (RA) to the DNA sample and mix by pipetting and gently flicking the tube, and spin down.

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP Beads (AXP).

Add 22 μl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking.

Incubate the reaction on a hula mixer (rotator mixer) for 10 minutes at room temperature.

Spin down the reaction and place it onto the magnet. Keep the tube on the magnet and pipette off the supernatant when it is clear and colourless.

Wash the beads by adding 125 μl of Long Fragment Buffer (LFB) and flick the beads to resuspend. Spin the reaction down and return it to the magnet and allow the beads to pellet. Remove and discard the supernatant when it is clear and colourless.

Repeat the previous step.

Spin the tube down and place it back on the magnet. Pipette off and discard any residual supernatant.

Remove the tube from the magnet and resuspend the pellet in 35 μl of Elution Buffer (EB). Spin down and incubate the reaction at 37°C for 10 minutes, occasionally flicking the tube to aid elution.

Spin the tube down and pellet the beads on a magnet for at least 1 minute, until the eluate is clear and colourless.

Remove and retain 35 μl of eluate containing the DNA library into a fresh 1.5 ml Eppendorf DNA LoBind tube.

Dispose of the pelleted beads.

CHECKPOINT

Quantify 1 μl of eluted sample using a Qubit fluorometer.

Expected Qubit measurements of ~28–30 ng/μl.

重要

We recommend loading 32 μl of your final prepared library onto the R10.4.1 Flow Cell.

We do not recommend diluting it with Elution Buffer (EB).

步骤结束

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

13. Priming and loading the SQK-APK114 library on the PromethION Flow Cell

材料
  • 测序缓冲液(SB)
  • 文库颗粒(LIB)
  • 文库溶液(LIS)
  • 测序芯片冲洗液(FCF)
  • Flush Tether UL (FTU)

耗材
  • PromethION 测序芯片
  • 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
重要

Ensure you have the SQK-APK114 MinKNOW script installed on to your sequencing device before preparing your flow cell and DNA library for sequencing.

For instructions on installing the SQK-APK114 script please see the Data acquisition and basecalling: Assembly Polishing Kit section of this protocol.

Note: This script will be pre-installed in future MinKNOW releases (MinKNOW 24.11 onwards).

Priming and loading the flow cell to sequence the Assembly Polishing Kit library

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.

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。

于室温下解冻测序缓冲液(SB)、文库颗粒(LIB)或文库溶液(LIS)、测序芯片系绳(FCT)和一管测序芯片冲洗液(FCF)。完全解冻后,涡旋振荡混匀,然后瞬时离心并置于冰上。 (1)

Prepare the flow cell priming mix in a suitable tube for the number of flow cells to flush. Once combined, mix well by briefly vortexing.

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

对PromethION 24/48,将测序芯片插入相应卡槽的对接端口:

  1. 将测序芯片与连接器横竖对齐,以便顺利卡入。

  2. 用力下压芯片至卡槽,并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

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。

Screenshot 2021-04-08 at 12.08.37

顺时针滑动加液孔孔盖,将其打开。

Prom Flowcell Loading 2 中文

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。

在加液孔打开的状态下,按下述步骤吸取少量液体,同时避免引入气泡:

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  3. 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。

Prom Flowcell Loading 3 中文

使用P1000移液枪向芯片的加液孔中加入500 µl芯片预处理溶液。加入过程中,请避免引入气泡。等待5分钟,与此同时,您可按以下步骤准备上样文库。

Prom Flowcell Loading 4 中文

将含有文库颗粒的LIB管用移液枪吹打混匀。

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)。

在一支新的1.5ml Eppendorf DNA LoBind离心管内,将所有试剂按以下顺序混合: (1)

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。

缓慢向芯片的加液口中加入500 µl预处理液,完成芯片的预处理。

Prom Flowcell Loading 5 中文

临上样前,用移液枪轻轻吹打混匀制备好的文库。

使用 P1000 移液枪向加液孔中加入200 µl 文库。

Prom Flowcell Loading 6 中文

合上加液孔孔盖。

Prom Flowcell Loading 7 中文

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。

如遮光片不在测序芯片上,请您按照以下步骤安装:

  1. 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。
  2. 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。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

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。

14. Data acquisition and basecalling: Assembly Polishing Kit

重要

Ensure you are using the most recent version of MinKNOW.

We recommend updating MinKNOW to the latest version prior to starting a sequencing run for the best sequencing results.

For more information on updating MinKNOW, please refer to our MinKNOW protocol.

重要

Installing the SQK-APK114 MinKNOW script

Please ensure you have the SQK-APK114 MinKNOW script installed on to your sequencing device.
This script will be pre-installed in future MinKNOW releases (MinKNOW 24.11 onwards).

1.Download the script (tar file) for your version of MinKNOW:

2. Extract the contents of the tar file. This will contain two files:

kits.toml
sequencing_PRO114_DNA_e8_2_260K_APK114.toml

3. Move the kits.toml to the directory /opt/ont/minknow/conf/package/sequencing/ on your device.

4. Move the sequencing_PRO114_DNA_e8_2_260K_APK114.toml to the directory /data/user_scripts/ on your device.

5. Open MinKNOW to the "Start sequencing" page.

6. Reload the scripts in the MinKNOW interface:

MinKNOW reload scripts T2T

7. SQK-APK114 will now appear as a selectable sequencing script in the MinKNOW UI.

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

We recommend first basecalling in real-time using the fast basecaller on MinKNOW using the PromethION 24 or 48 device. MinKNOW can be used and set up to sequence in multiple ways:

  • On a computer either directly or remotely connected to a sequencing device.
  • Directly on a PromethION 24/48 sequencing device.

After real-time basecalling, rebasecall the data using the assembly polishing model in Dorado.

For more information on using MinKNOW on a sequencing device, please see the PromethION 24/48 user manual.


Real-time sequencing

To start a run on MinKNOW to sequence the Assembly Polishing Kit library:

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Assembly Polishing Kit V14 (SQK-APK114) on the Kit page

4.

Configure the sequencing parameters as follows:
Basecalling: off
Modified bases: off
Model: N/A
Barcoding: off
Alignment: off
Adaptive sampling: off
Advanced options: default settings

5.

Configure the data targets as follows:
Run targets: Run limit
Action: Stop run when
Condition: Flow cell is
Value: End of life

6.

Configure the analysis workflow:
Workflow: off

7.

Configure the output parameters as follows:
Basecalled output type: .BAM
Based on: Time elapsed
Frequency: Every 10 minutes
ASTQ options - Compression: on
Raw reads: on
POD5: on
FAST5: off

8.

Configure the filterning options as follows:
Filtering: on
Min Qscore: 10
Min read length (kb): 1

Post-sequencing basecalling

1. Ensure you have installed Dorado ≥0.7.2 from the installation section of the Dorado Github page.

2. Download the the custom assembly polishing command and the basecalling model, and rebasecall your data as described on the Dorado Github page by using the commands outlined below:

$ dorado download dna_r10.4.1_e8.2_apk_sup@v5.0.0

$ dorado basecaller --min-qscore 10 dna_r10.4.1_e8.2_apk_sup@v5.0.0 pod5s/ > apk_reads.bam

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

In the Downstream analysis section, we outline further options for analysing your basecalled data for the telomere-to-telomore experiment.

15. Sample preparation: custom SPRI bead preparation

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
  • 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
  • 盛有冰的冰桶

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 before library preparation.

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

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.

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.

16. Sample preparation: whole blood cell isolation

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
  • Centrifuge with capacity for 5 ml and 15 ml tubes, and a swing out and fixed angle rotors
  • 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
  • 盛有冰的冰桶
可选仪器
  • Liquid nitrogen and canister
  • -80°C freezer storage

White blood cells isolation for the Pore-C DNA experiment

Before starting the Pore-C DNA extraction, the white blood cells must be isolated from whole blood whilst maintaining cell viability. Approximately 5–10 ml of whole blood should yield sufficient white blood cells 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 white blood cells are prepared in aliquots of 1x white blood cells supplemented with 2% FBS. Approximately 10 million PBMCs are taken forwards into the Pore-C sample preparation step.

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

  • white blood cells are isolated as soon as possible from fresh blood and no later than 24 hours.
  • white blood cells 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.

Prepare three solutions in preparation for white blood cells isolation:

  • 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

Allow the whole blood sample to warm to room temperature and then dilute with equal volume of room temperature 1X PBS supplemented with 2% FBS. Transfer the diluted blood to a 50 ml centrifuge tube.

Centrifuge at 800 x g at 20°C for 10 minutes with the brake off to prevent remixing of the separated fractions.

After centrifugation, the whole blood should have separated into the plasma, buffy coat and red blood cells. Check the turbidity of the plasma layer (the top layer). If it is not clear, centrifuge at 800 x g at 20°C for a further 10 minutes with the brake off.

blood Pore-C diagram

Using a Pasteur pipette, remove as much of the plasma layer as possible without disturbing the layer of buffy coat. Gently remove the buffy coat layer, taking care to draw as little of the red blood cell layer as possible. Transfer the recovered buffy coat to a fresh 50 ml centrifuge tube.

Make up the recovered buffy coat sample to 25 ml of 1X PBS supplemented with 2% FBS.

Aliquot 20 ml of 1X PBS supplemented with 60% Percoll in a fresh 50 ml centrifuge tube.

Using a fresh Pasteur pipette, very gently layer the diluted buffy coat sample over the Percoll layer at a 45° angle.

45 angle

Centrifuge at 350 x g at 20°C for 40 minutes with slow acceleration and with the brake off.

Check the turbidity of the plasma layer and the formation of the white blood cells layer. If the plasma layer is not clear or the PBMC layer is not well defined, continue to centrifuge at 350 x g at 20°C for a further 20 minutes using slow acceleration with the brake off.

blood Pore-C diagram2

Using a Pasteur pipette, remove as much of the plasma layer as possible without disturbing the layer of white blood cells, then gently remove the layer of PBMCs. It is acceptable to draw plasma with the layer of white blood cells; however, take care to draw as little of the Percoll layer as possible.

Transfer the recovered white blood cells to a fresh 50 ml centrifuge tube.

Resuspend the recovered white blood cells in 50 ml of room temperature 1X PBS supplemented with 2% FBS.

Centrifuge at 350 x g at 20°C for 15 minutes with the brake on.

Aspirate and discard the supernatant. Gently resuspend the white blood cells in 25 ml of room temperature 1X PBS supplemented with 2% FBS. Centrifuge at 350 x g at 20°C for 15 minutes with the brake on.

Repeat the previous step.

Aspirate and discard the supernatant. Gently resuspend the white blood cells in another 25 ml of room temperature 1X PBS supplemented with 2% FBS.

Centrifuge at 200 x g at 20°C for 10 minutes with the brake on.

Assuming every 1 ml of whole blood originally used will yield approximately 1.5 million white blood cells, resuspend cells to approximately 10 million white blood cells/ml in room temperature 1X PBS supplemented with 2% FBS.

Transfer an aliquot of approximately 10 million white blood cells total to a fresh 2 ml Eppendorf DNA LoBind tube.

Cool on ice for 5 minutes.

可选操作

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

  1. Centrifuge at 350 x g at 4°C for 2 minutes with the brake on.
  2. Aspirate and discard the supernatant, then resuspend the white blood cells pellet in 1 ml of chilled FBS.
  3. 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.
  4. 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.

17. Sample preparation: Pore-C extraction

材料
  • 10 million white blood cells isolated from whole blood
  • Custom SPRI bead suspension

耗材
  • Qubit dsDNA HS Assay(双链DNA高灵敏度检测)试剂盒(ThermoFisher,Q32851)
  • 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)
  • T4 DNA Ligase 400,000 U/ml (NEB, M0202S/L)
  • 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)
  • Chilled 10X phosphate-buffered saline (PBS) (Thermo Fisher, 70011044)
  • Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
  • Recombinant Albumin at 20 μg/μl (NEB, B9200S)
  • 5 M NaCl (Sigma, 71386)
  • 3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
  • Tween-20 (Thermo Scientific, J20605.AP)
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • Proteinase K at 20 μg/μl (NEB, P8107S)
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
  • Ethanol, 100% (e.g. Fisher, 16606002)
  • 新制备的80%乙醇(用无核酸酶水配制)
  • Nuclease-free water
  • Ziplock bags
  • 0.2 µm filter
  • 50 ml centrifuge tubes
  • 15 ml Falcon tubes
  • 5 ml centrifuge tubes
  • 2 ml Eppendorf DNA LoBind 离心管
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Qubit™ 分析管(Invitrogen, Q32856)

仪器
  • Class I hood with active charcoal filter
  • 迷你离心机
  • Temperature-controlled centrifuge
  • Thermal cycler or heat block
  • Thermomixer
  • Temperature-controlled microfuge
  • Vortex mixer
  • Hula混匀仪(低速旋转式混匀仪)
  • Qubit荧光计 (或用于质控检测的等效仪器)
  • 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
  • 盛有冰的冰桶
  • -80°C freezer storage

Pore-C extraction

Pore-C extraction is performed across three days to stabilise the three-dimensional interactions of the DNA in the nucleus before extracting the DNA.

Day 1

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 the NlaIII restriction enzyme and CutSmart Buffer in accordance with the manufacturer's instructions and place on ice.

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

Prepare 1 ml of 1% SDS in nuclease-free water, as follows:

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

Prepare 10 ml of 10% (v/v) ECOSURF™ EH-9 in nuclease-free water, as follows:

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

Prepare 10 ml of 10% (v/v) IGEPAL CA-630 in nuclease-free water, as follows:

Note: We recommend using a wide-bore pipette tips when handling the IGEPAL CA-630.

  1. Add 1 ml ofIGEPAL CA-630 to a fresh Falcon tube.
  2. Add 9 ml of nuclease-free water to the same tube.
  3. Gently pipette mix with a wide-bore pipette tip until the solution is homogenous.

Prepare 1 ml of 2.5 M glycine filtered through a 0.2 µm filter and store at room temperature.

Prepare 200 ml filtered 1X PBS and chill at 4°C.

Pre-cool a centrifuge to 4°C.

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.

Prepare the formaldehyde solution as follows:

  1. Transfer 10 ml of 1X PBS into a 50 ml Falcon tube. Note: Using a 15 ml Falcon tube is not recommended.

  2. 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.

  3. Mix by gentle inversion, and open the tube to allow gases to escape, then close the tube.

  4. Check that no formaldehyde residue has remained on the gloves, Falcon tube, or pipette.

  5. Remove the outer gloves and discard them in a biohazard bag in the hood.

  6. Remove the 1% formaldehyde 1X PBS solution from the hood.

  7. Store the tube with formaldehyde inside a zip lock bag at 4°C prior to use.

Prepare the white blood cells as follows:

  1. Take approximately 10 million white blood cells and briefly homogenise the suspension by gently pipetting with a wide-bore pipette tip.

  2. Transfer the cell suspension to a 50 ml centrifuge tube.

  3. Rinse the original tube with a further 1 ml of chilled 1X PBS into the 50 ml centrifuge tube.

  4. Bring the volume of the resuspended white blood cells to 10 ml in chilled 1X PBS.

  5. Proceed with the Pore-C experiment using approximately 10 million white blood cells as input.

Centrifuge the sample at 300 x g at 4°C for 5 minutes.

Aspirate and discard the supernatant, then add 10 ml of chilled 1X PBS to the pellet. Resuspend the pellet by gently pipetting up and down using a wide-bore pipette tip.

Centrifuge the sample at 300 x g at 4°C for 5 minutes.

Check the 2.5 M glycine solution has not precipitated before crosslinking the sample. Dissolve precipitate with heat and vortexing if required.

Inside a class I hood, with double gloves, aspirate and discard the supernatant.

Add 1 ml of the previously prepared 1% formaldehyde solution 1X PBS to the pellet. Resuspend the pellet by gently pipetting up and down using a wide-bore pipette tip.

Once resuspended, add a further 9 ml of the 1% formaldehyde solution in 1X PBS. Mix gently by pipetting up and down, using a wide-bore pipette tip.

Incubate at room temperature for exactly 10 minutes to crosslink the sample. The incubated solution should be mixed by gentle agitation every few minutes.

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.

Inside the hood with double gloves, quench the formaldehyde by adding 527 μl of 2.5 M glycine to the sample suspension for a final concentration of 1% w/v glycine (125 mM) in ~10.5 ml. Mix gently by pipetting up and down, using a wide-bore pipette tip.

Incubate at room temperature for 5 minutes, then chill on ice for a further 10 minutes with regular, gentle agitation.

Centrifuge the crosslinked sample suspension at 300 x g at 4°C for 5 minutes.

Continuing in the class I hood, aspirate and discard the supernatant. Add 10 ml of chilled 1X PBS to the tube.

Centrifuge the sample at 500 x g at 4°C for 5 minutes.

Continuing in the class I hood, aspirate and discard the supernatant, and add 1 ml of chilled 1X PBS to the pellet. Mix gently by pipetting up and down using a wide-bore pipette tip.

Split the resuspended sample into two separate 500 μl aliquots in fresh 2 ml Eppendorf tubes.

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

Wash the previous sample tube with a further 1 ml of 1X PBS, and split this between the two aliquots in 2 ml Eppendorf DNA LoBind tubes.

提示

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

Centrifuge the samples at 500 x g at 4°C for 5 minutes. Aspirate and discard the supernatant.

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.

Pre-cool a microfuge to 4°C and set a thermomixer to 65°C.

Prepare 600 μl of 1.5X CutSmart Buffer in nuclease-free water as follows in a 1.5 ml Eppendorf DNA LoBind tube. Keep on ice.

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

To make the permeabilisation solution, add the components below to a 1.5 ml Eppendorf DNA LoBind tube in the following order. Keep the prepared permeabilisation solution on ice at 4°C until ready to use.

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

Thaw the protease inhibitor cocktail on ice and spin down.

Add 50 μl of protease inhibitor cocktail to 500 μl of permeabilisation solution at 4°C.

Add 550 μl protease inhibitor cocktail-permeabilisation solution to the sample pellet. Resuspend the pellet by gently pipetting up and down, using a wide-bore pipette tip.

Incubate on ice for 15 minutes and mix by regular, gentle inversion.

Centrifuge the sample at 500 x g at 4°C for 10 minutes.

重要

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

Resuspend the pellet in 200 μl of the prepared chilled 1.5X CutSmart buffer by gently pipetting up and down, using a wide-bore pipette tip.

Centrifuge the sample at 500 x g at 4°C for 5 minutes. Aspirate and discard the supernatant.

Resuspend the pellet in 300 μl of the prepared chilled 1.5X CutSmart buffer by gently pipetting up and down, using a wide-bore pipette tip.

To denature the chromatin, add 33.5 μl 1% SDS directly to the sample suspension to a final concentration of 0.1% SDS and a total volume of 333.5 μl. Mix gently by pipetting up and down using a wide-bore pipette tip.

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

Incubate the sample suspension in a thermomixer at 300 RPM at 65°C for 10 minutes.

Note: This incubation can be performed without mixing.

Remove the tube from the thermomixer and immediately put on ice.

Set the thermomixer to 37°C.

Add 37.5 μl of 10% (v/v) ECOSURF™ EH-9 directly to the cell suspension for a final concentration of 1% ECOSURF™ EH-9 (total volume of 371 μl). Mix gently by pipetting with a wide-bore pipette tip.

Incubate the tube on ice for 10 minutes.

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

Add the following reagents to the sample suspension and invert 3-4 times to mix.

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

Incubate the tube in a thermomixer at 37°C for 18 hours with periodic <1000 rpm rotation for <30 seconds every 15 minutes. This will prevent condensation inside the lid.

Note: This incubation can be performed without mixing.

重要

During the long/overnight incubation step, please ensure all your reagents are stored appropriately until the incubation has finished by following the manufacturers recommendations.

Day 2

During day 2, 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 T4 DNA Ligase and T4 DNA Ligase Reaction Buffer in accordance with the manufacturer's instructions and place on ice.

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

Prepare 5 ml of 20% Tween-20 in nuclease free water as follows:

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

Set the thermomixer to 65°C.

Heat denature the restriction enzyme by incubating the sample suspension in the thermomixer at 65°C with 300 rpm rotation for 20 minutes. Allow the reaction to cool to room temperature.

Set the thermomixer to 16°C.

Set up the proximity ligation reaction according to the table below, adding reagents directly to the sample suspension in the following order. Mix gently by pipetting up and down, using a wide-bore pipette tip.

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

Incubate the sample suspension in a thermomixer at 16°C for 6 hours, with periodic <1000 RPM rotation for <30 seconds every 15 minutes. This prevents condensation inside the lid.

Note: This incubation can be performed without mixing.

重要

Do not extend the 6-hour incubation as prolonged ligation may increase trans-chromosomal contacts in the Pore-C data.

Set the thermomixer to 56°C.

Add the reagents to the previous ligation reaction in the following order to make up the protein degradation reaction. Mix the sample gently by inverting the tube 3–4 times.

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

Incubate the sample suspension in a thermomixer at 56°C for 18 hours with periodic <1000 rpm rotation for <30 seconds every 15 minutes to prevent condensation inside the lid.

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.

重要

We do NOT recommend performing this overnight incubation at higher temperatures as enzyme activity will reduce over time.

Day 3

During day 3, 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.

Pre-cool the centrifuge to 15°C.

Place the sample on ice until cool, then transfer the entire volume to a 5 ml centrifuge tube.

Rinse the original tube with a further 200 μl of nuclease-free water and add this to the same 5 ml centrifuge tube for a total sample volume of ~2200 μl.

Add an equal volume of chilled phenol:chloroform:isoamyl alcohol 25:24:1 saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA, adjusting this volume as needed to match that of the sample. Mix by gently inverting the tube for 5 minutes to achieve a homogeneous emulsion.

Centrifuge the aliquots at 16,000 x g at 15°C for 15 minutes.

Incubate the aliquots on ice for 2 minutes until the organic phase becomes cloudy; this will strengthen the integrity of the interphase layer.

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.

Transfer the aqueous phase into a fresh 5 ml centrifuge tube for each aliquot and make note of the recovered volume (expect ~2,000 μl).

Transfer half of the recovered aqueous phase to a second 5 ml centrifuge tube to create two equal aliquots.

For each aliquot, add 0.02X of 5 M NaCl (0.1 M final) and 0.1X of 3 M sodium acetate pH 5.5 (0.3 M final), relative to the volume of the recovered aqueous phase of the aliquot. Mix by gently inverting the tube. (1)

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

For example, for a total recovered volume of 2,000 µl, to each 1,000 µl aliquot add:

  • 20 µl of 5 M NaCl
  • 100 µl of 3 M sodium acetate

For each aliquot, add 3X of 100% ethanol relative to the volume of the recovered aqueous phase. Mix by gently inverting the tubes.

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

Precipitate at –80°C for >1 hour.

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.

Pre-cool a centrifuge to 4°C.

Centrifuge the sample at 16,000 x g at 4°C for 30 minutes.

Aspirate and discard the supernatant, then wash the pellets with 4 ml of 80% ethanol.

Centrifuge the sample at 16,000 x g at 4°C for 5 minutes.

Aspirate and discard the supernatant, then wash the pellets with 2 ml of 70% ethanol.

Centrifuge the sample at 16,000 x g at 4°C for 5 minutes.

Aspirate and discard the supernatant. Briefly spin down the tubes and aspirate any residual supernatant. Allow the pellets to dry for 5 minutes.

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

Carefully resuspend each aliquot in 75 μl of TE buffer. Incubate for 5 minutes at room temperature, mixing by gently inverting the tube every few minutes.

Briefly spin down the tubes, then transfer and pool all aliquots together into a 1.5 ml Eppendorf DNA LoBind 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.

Dilute your sample to 60 ng/µl in a final volume of 50 µl of TE buffer at pH 8.

Add 42.5 µl (0.85X) of room temperature custom SPRI bead suspension and mix by flicking the tube.

将离心管置于Hula混匀仪(低速旋转式混匀仪)上室温孵育10分钟。

Spin down briefly and pellet on a magnet until the supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.

保持离心管在磁力架上不动,以200µl新鲜制备的80%乙醇洗涤磁珠。小心不要扰动磁珠。用移液枪将乙醇吸走并弃掉。

如在此过程中不慎扰动磁珠,请静待磁珠和液相分离后再吸出乙醇。

重复上述步骤。

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的乙醇。让磁珠在空气中干燥约30秒,但不要干至表面开裂。

Remove the tube from the magnetic rack and resuspend the pellet in 40 µl of TE buffer. Incubate for 1 minute at 50°C, and then for 5 minutes at room temperature.

将离心管静置于磁力架上至少一分钟,直到磁珠和液相分离,且洗脱液澄清无色。

Remove and retain 40 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

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

Take forwards 2 µg of Pore-C DNA extract into the next step. Store the DNA at 4°C until the next step can begin.

18. Library preparation: Pore-C sequencing

材料
  • 2 μg Pore-C DNA extract
  • 连接测序试剂盒V14(SQK-LSK114)

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
  • 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
  • Qubit荧光计 (或用于质控检测的等效仪器)
  • 盛有冰的冰桶
  • P1000移液枪和枪头
  • 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

Library preparation for the Pore-C experiment

In this step, the extracted Pore-C DNA is prepared for sequencing by repairing any nicks in the DNA and preparing the ends for sequencing adapter attachment.

根据生产厂家的说明准备NEBNext FFPE DNA 修复混合液和 NEBNext Ultra II 末端修复/ dA尾添加模块,并置于冰上。

为获得最优表现,NEB建议如下:

  1. 于冰上解冻所有试剂。

  2. 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。

  3. 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。

  4. 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。

  5. FFPE DNA 修复缓冲液可能轻微泛黄,不影响使用。

Prepare the DNA in nuclease-free water:

  1. Transfer 2 μg input DNA into a 1.5 ml Eppendorf DNA LoBind tube.
  2. Adjust the volume to 47 μl with nuclease-free water.
  3. Mix thoroughly by pipetting up and down, or by flicking the tube.
  4. Spin down briefly in a microfuge

在一支0.2ml的薄壁PCR管中,混合以下试剂:

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

轻轻吹打以充分混匀,并瞬时离心。

Using a thermal cycler, incubate at 20°C for 15 minutes and 65°C for 5 minutes.

Resuspend the AMPure XP Beads by vortexing.

将DNA样本转至干净的1.5 ml Eppendorf DNA LoBind离心管中。

Add 60 µl of resuspended the AMPure XP Beads to the end-prep reaction and mix by flicking the tube.

将离心管置于Hula混匀仪(低速旋转式混匀仪)上室温孵育5分钟。

准备500μl新制备的80%乙醇(用无核酸酶水配制)。

将样品瞬时离心,并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动,用移液枪吸去清液。

保持离心管在磁力架上不动,以200µl新鲜制备的80%乙醇洗涤磁珠。小心不要吹散磁珠。用移液枪将乙醇吸走并弃掉。

重复上述步骤。

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的乙醇。让磁珠在空气中干燥约30秒,但不要干至表面开裂。

将离心管从磁力架上移开。将磁珠重悬于61µl无核酸酶的水中。室温下孵育2分钟。

将离心管静置于磁力架上至少一分钟,直到磁珠和液相分离,且洗脱液澄清无色。

将61µl洗脱液转移至一支新的1.5ml Eppendorf DNA LoBind管中。

CHECKPOINT

取1µl洗脱样品,用Qubit荧光计定量。

提示

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。

瞬时离心连接接头(LA)和快速T4 DNA连接酶,置于冰上。

于室温下解冻连接缓冲液(LNB),解冻后瞬时离心,并用移液枪吹打混匀。该缓冲液的黏度较高,涡旋振荡会很难混匀。解冻并混匀后,请立即置于冰上。

将洗脱缓冲液(EB)于室温下解冻,涡旋振荡混匀后,再瞬时离心,置于冰上。

Thaw the Short Fragment Buffer (SFB) at room temperature and mix by vortexing. Then spin down and place on ice.

在一支1.5ml Eppendorf DNA LoBind离心管内,将所有试剂按以下顺序混合: (1)

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

轻轻吹打以充分混匀,并瞬时离心。

室温下孵育10分钟。

Resuspend the AMPure XP Beads by vortexing.

Add 40 µl of resuspended AMPure XP Beads to the reaction and mix by flicking the tube.

将离心管置于Hula混匀仪(低速旋转式混匀仪)上室温孵育5分钟。

将样品瞬时离心,并静置于磁力架上待磁珠和液相分离。保持离心管在磁力架上不动,用移液枪吸去上清液。

Wash the beads by adding 250 μl Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

重复上述步骤。

将离心管瞬时离心后置于磁力架上。用移液枪吸走残留的上清液。让磁珠在空气中干燥约30秒,但不要干至表面开裂。

将离心管从磁力架上移开。将磁珠重悬于25µl洗脱缓冲液中(EB)。瞬时离心,然后在室温下孵育10分钟。对于高分子量的DNA,在37°C下孵育可以提高长片段的回收率。

将离心管静置于磁力架上至少一分钟,直到磁珠和液相分离,且洗脱液澄清无色。

将此含有DNA文库的25µl洗脱液转移至一支新的1.5ml Eppendorf DNA LoBind管中。

弃掉磁珠。

CHECKPOINT

取1µl洗脱样品,用Qubit荧光计定量。

In a fresh 1.5 ml Eppendorf DNA LoBind tube, make up your DNA library for Flow Cell loading to 32 µl using Elution Buffer (EB) as follows:

Reagent Volume
Eluted DNA sample, from the previous step 5 µl
Elution Buffer (EB) 27 µl
Total 32 µl
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. Priming and loading Pore-C library on the PromethION Flow Cell

材料
  • 测序缓冲液(SB)
  • 文库颗粒(LIB)
  • 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

Pore-C experiment flow cell loading

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

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。

于室温下解冻测序缓冲液(SB)、文库颗粒(LIB)或文库溶液(LIS)、测序芯片系绳(FCT)和一管测序芯片冲洗液(FCF)。完全解冻后,涡旋振荡混匀,然后瞬时离心并置于冰上。 (1)

Prepare the flow cell priming mix in a suitable tube for the number of flow cells to flush. Once combined, mix well by briefly vortexing.

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

对PromethION 24/48,将测序芯片插入相应卡槽的对接端口:

  1. 将测序芯片与连接器横竖对齐,以便顺利卡入。

  2. 用力下压芯片至卡槽,并确认卡夹位置归位。

Prom Flowcell Loading 1a 中文

Prom Flowcell Loading 1b 中文

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。

Screenshot 2021-04-08 at 12.08.37

顺时针滑动加液孔孔盖,将其打开。

Prom Flowcell Loading 2 中文

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。

在加液孔打开的状态下,按下述步骤吸取少量液体,同时避免引入气泡:

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入加液孔中。
  3. 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。

Prom Flowcell Loading 3 中文

使用P1000移液枪向芯片的加液孔中加入500 µl芯片预处理溶液。加入过程中,请避免引入气泡。等待5分钟,与此同时,您可按以下步骤准备上样文库。

Prom Flowcell Loading 4 中文

将含有文库颗粒的LIB管用移液枪吹打混匀。

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)。

在一支新的1.5ml Eppendorf DNA LoBind离心管内,将所有试剂按以下顺序混合: (1)

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。

缓慢向芯片的加液口中加入500 µl预处理液,完成芯片的预处理。

Prom Flowcell Loading 5 中文

临上样前,用移液枪轻轻吹打混匀制备好的文库。

使用 P1000 移液枪向加液孔中加入200 µl 文库。

Prom Flowcell Loading 6 中文

合上加液孔孔盖。

Prom Flowcell Loading 7 中文

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。

如遮光片不在测序芯片上,请您按照以下步骤安装:

  1. 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。
  2. 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。

Prom Flowcell Loading 8a 中文

Prom Flowcell Loading 8b 中文

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. Data acquisition and basecalling: Pore-C

重要

Ensure you are using the most recent version of MinKNOW.

We recommend updating MinKNOW to the latest version prior to starting a sequencing run for the best sequencing results.

For more information on updating MinKNOW, please refer to our MinKNOW protocol.

How to start sequencing

Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.

We recommend basecalling in real-time using the high-accuracy (HAC) basecaller on MinKNOW using the PromethION 24 or 48 device. MinKNOW can be used and set up to sequence in multiple ways:

  • On a computer either directly or remotely connected to a sequencing device.
  • Directly on a PromethION 24/48 sequencing device.

For more information on using MinKNOW on a sequencing device, please see the PromethION 24/48 user manual.


Real-time sequencing

To start a run on MinKNOW to sequence Pore-C DNA

1. Navigate to the start page and click Start sequencing.

2. Fill in your experiment details, such as name and PromethION Flow Cell position and sample ID.

3. Select the Ligation Sequencing V14 (SQK-LSK114) on the Kit page.

4.

Configure the sequencing parameters as follows:
Basecalling: on
Modified bases: off
Model: High-accuracy (HAC) basecalling
Barcoding: off
Alignment: off
Adaptive sampling: off
Advanced options: default settings

5.

Configure the data targets as follows:
Run duration: 72 hours

6.

Configure the analysis workflow:
Workflow: off

7.

Configure the output parameters as follows:
Basecalled output type: .BAM & .FASTQ
Based on: Time elapsed
Frequency: Every 10 minutes
FASTQ options - Compression: on
Raw reads: on
POD5: on
FAST5: off

8.

Configure the filterning options as follows:
Filtering: on
Min Qscore: 9
Min read length (kb): 0.2

Data analysis after sequencing

Your Pore-C experiment data is basecalled live in MinKNOW during sequencing, using the high-accuracy (HAC) basecaller.

In the Downstream analysis section, we outline further options for analysing your basecalled data for the telomere-to-telomore experiment.

21. Downstream analysis

重要

Telomere-to-telomere post-basecalling analysis compute requirements:

Post-basecalling analysis compute requirements:
· A100 Data Acquisition Unit
or
· AWS ec2 “g5.24xlarge” instance for read correction and polishing (Dorado and Medaka) and "x1.32xlarge" instance for assembly (Verkko)

Do NOT perform downstream analysis on your A100 Data Acquisition Unit while it is being used for live sequencing
Running analysis on an A100 Data Acquisition Unit during live sequencing may interfere with data acquisition and/or cause software failures.

Telomere-to-telomere assembly

Assembly workflow

To carry out the telomere-to-telomere downstream analysis, we recommend previous bioinformatics experience. The commands below combine all three datasets from the ultra-long DNA, Pore-C, and Assembly Polishing Kit experiments.

Workflow analysis t2t pic

More information about the workflow and examples of the inputs and outputs of this analysis protocol can be found on the nanopore-only telomere-to-telomere dataset and blog post on the EPI2ME page.

An EPI2ME workflow combining all three datasets will be released in the future to give more accessibility for users of all experience. More information about this release will be published on the Community.

Note: all basecalling must have been completed before starting downstream analysis.



Setting up the environment

The Nanopore-only telomere-to-telomere assembly workflow is summarised in the figure above and requires the following tools and their dependencies to be installed:

  • Dorado (≥v0.7.3) for read correction
  • Verkko (= v2.1) for assembly
  • Medaka (= v1.12.1) for polishing
  • Minimap2 (≥v2.27) for alignment
  • Samtools (≥v1.16) for format conversion

See the appendix below for an example how to set up a suitable environment. For Dorado, please follow installation instructions on the Github page.

For more information about the workflow and for examples of the inputs and outputs of this analysis protocol, please see the Nanopore-only T2T dataset. The basecalled data from s3://ont-open-data/londoncalling2024/assembly/basecalling/ may be downloaded to test the full workflow. Instructions to download the dataset from S3 are outlined in the EPI2ME blog post.



Read filtering

Basecalled reads for your Ultra-long DNA experiment (unaligned .bam) should be filtered for reads with mean quality of ≥ 10. We also recommend filtering for ≥ 10kb read length to speed up run time but this is optional.

Dorado reports read mean quality as a “qs” tag in the output .bam file. The command to filter ULK reads by quality and read length is:

samtools view -@ <threads> -b \ 
 -e '[qs] >= 10 && length(seq) > 10000' \ 
 input.bam > output.bam  

Note: If not already done, the command to filter APK and Pore-C reads by quality is:

samtools view -@ <threads> -b -e '[qs] >= 10' input.bam > output.bam 



Ultra long read correction

First, pass the basecalled Ultra Long (SQK-ULK114) reads through read correction in Dorado.

To do this, the basecalled Ultra Long BAMs should first be combined into a single BAM:

samtools merge -@ <threads> -o ulk_reads.bam \ 
  ulk_reads_flowcell_1.bam ulk_reads_flowcell_2.bam 

This merged ULK BAM should then be converted to fastq:

samtools fastq -@ <threads> \ 
 ulk_reads.bam > uncorrected_ulk_reads.fastq 

Then passed through Dorado read correction:

dorado correct uncorrected_ulk_reads.fastq > corrected_ulk_reads.fasta 



Assembly instructions

1. Assemble the corrected reads using the Verkko assembler.

1.1. The basecalled Pore-C BAM should also be converted to fastq for this step:

samtools fastq -@ <threads> porec_reads.bam > porec_reads.fastq 

1.2. Run Verkko using the following command :

verkko –d asm \ 
--hifi corrected_ulk_reads.fasta \ 
--nano uncorrected_ulk_reads.fastq \  
--porec porec_reads.fastq \
--no-correction

2. Having run Verkko in its entirety, the last step is to polish the Verkko assembly (assembly.fasta) using data from the APK sequencing kit.
This is done using the medaka_consensus_joint script which simultaneously uses both ULK and APK data for error correction and will be in the path in the Medaka environment.

medaka_consensus_joint \ 
   -i "${APK_BAM}" -v apk -i "${ULK_BAM}" -v ulk \ 
   -t ${THREADS} -o "${OUTPUT}" \ 
   -m r1041_e82_260bps_joint_apk_ulk_v5.0.0 \ 
   -d "${VERKKO_ASSEMBLY}" 



Appendix

Example: Installing required software with conda

0. Install conda (or mamba, micromamba) if you do not already have it.
1. Add the following to a file called t2t.yml
name: t2t
channels:
- conda-forge
- bioconda
- nvidia
dependencies:
- python=3.9
- verkko=2.1
- minimap2>=2.27
- samtools>=1.16
- libcublas
- bcftools>=1.16
- pip
- pip:
- medaka == 1.12.1
2. To create the environment:
conda env create --file=t2t.yml
3. To activate the environment:
conda activate t2t

22. 测序芯片的重复利用及回收

材料
  • 测序芯片清洗剂盒(EXP-WSH004)

完成测序实验后,如您希望再次使用测序芯片,请按照测序芯片清洗试剂盒的说明进行操作,并将清洗后的芯片置于2-8℃保存。

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。

请按照“回收程序”清洗好芯片,以便送回Oxford Nanopore。

您可在 此处找到回收测序芯片的说明。

请注意: 在将测序芯片寄回之前,请使用去离子水对每张芯片进行冲洗。

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。

23. 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。 SPRI cleanup
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。

24. 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。DNA gel2 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)

image2022-3-25 10-43-25 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 MK1B温度控制的更多信息,请参考此 FAQ (常见问题)文档。

Last updated: 11/1/2024

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