Ligation sequencing gDNA - exome enrichment (SQK-LSK110)

Oxford Nanopore Technologies
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MinION: Protocol

Ligation sequencing gDNA - exome enrichment (SQK-LSK110) V EXS_9118_v110_revI_10Nov2020

For enrichment of a specific target region for:

  • Highest yield
  • Depth of coverage of region
  • Larger number of target regions
  • If amplification is impractical
  • Compatible with R10.3 flow cells

For Research Use Only

This is a Legacy product This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

FOR RESEARCH USE ONLY

概览

For enrichment of a specific target region for:

  • Highest yield
  • Depth of coverage of region
  • Larger number of target regions
  • If amplification is impractical
  • Compatible with R10.3 flow cells

For Research Use Only

This is a Legacy product This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com.

Document version: EXS_9118_v110_revI_10Nov2020

1. Overview of the protocol

重要

This is a Legacy product

This kit is soon to be discontinued and we recommend all customers to upgrade to the latest chemistry for their relevant kit which is available on the Store. If customers require further support for any ongoing critical experiments using a Legacy product, please contact Customer Support via email: support@nanoporetech.com. For further information on please see the product update page.

Exome sequencing protocol features

Use this protocol if you:

  • Are not interested in analysing the entire genome
  • Want greater depth of coverage of a specific exonic region
  • Want to analyse a large number of target regions

Introduction to exome sequencing

This protocol describes how to carry out exome sequencing using the Ligation Sequencing Kit (SQK-LSK110) and the Agilent SureSelect method.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Ensure you have your sequencing kit, the correct equipment and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Library preparation

You will need to:

  • Fragment your DNA
  • Ligate PCR adapters to the DNA ends and amplify the fragments
  • Hybridise the DNA to probes provided in the Agilent SureSelect Exome kit, and perform a pulldown
  • Elute and amplify the pulled-down fragments
  • Prepare the DNA ends for adapter attachment
  • Attach sequencing adapters supplied in the kit to the DNA ends
  • Prime the flow cell, and load your DNA library into the flow cell

Exome sequencing

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads
  • Start the EPI2ME software and select a workflow for Human Exome mapping, or use minimap2 to align the reads to the human reference genome.

We recommend using the standard SureSelect Target Enrichment System protocol from Agilent Technologies with the following differences:

  • The DNA in this protocol is sheared with dsDNA Fragmentase rather than in a Covaris g-TUBE
  • DNA purification in this protocol is carried out using Agencourt AMPure XP beads instead of QIAquick PCR purification
  • DNA End-prep is carried out using the NEBNext Ultra II End Repair/dA-Tailing Module instead of the Agilent method
  • Ligation of PCR adapters is carried out using the NEB Blunt/TA Ligase Master Mix instead of the Agilent method
  • No desalting of the capture solution is necessary
  • LongAMP Taq is used for amplification of the DNA post-hybridisation
重要

This protocol is for the preparation of a single sample. Multiple samples can be prepared in parallel; each sample will require a separate pull-down and will need to be run on a separate flow cell.

重要

Compatibility of this protocol

This protocol should only be used in combination with:

  • Ligation Sequencing Kit (SQK-LSK110)
  • Control Expansion (EXP-CTL001)
  • PCR Expansion (EXP-PCA001)
  • FLO-MIN106 (R9.4.1) flow cells
  • Flow Cell Wash Kit (EXP-WSH004)

2. Equipment and consumables

材料
  • 3 µg high molecular weight human genomic DNA
  • Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
  • Custom primer mix, 10 μM (IDT) - see below for sequences
  • Ligation Sequencing Kit (SQK-LSK110)
  • PCR Expansion (EXP-PCA001)
耗材
  • NEBNext dsDNA Fragmentase (M0348L)
  • NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
  • NEBNext Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • NEBNext 快速连接模块（NEB，E6056）
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • Cot-1 DNA (ThermoFisher Scientific 15279-011)
  • Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
  • Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'
  • LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 新制备的70%乙醇（用无核酸酶水配制）
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
  • 0.2 ml 96 well PCR plate
仪器
  • Hula混匀仪（低速旋转式混匀仪）
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • 迷你离心机
  • 涡旋混匀仪
  • 热循环仪
  • 盛有冰的冰桶
  • 计时器
  • SpeedVac
  • 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
  • P2移液枪和枪头
可选仪器
  • Standard gel electrophoresis equipment
  • Agilent生物分析仪（或等效仪器）
  • Qubit fluorometer (or equivalent for QC check)
  • Eppendorf 5424 离心机（或等效器材）

For this protocol, you will need 3 µg high molecular weight human genomic DNA.

起始DNA

DNA质控

选择符合质量和浓度要求的起始DNA至关重要的。使用过少或过多的DNA，或者质量较差的DNA（如，高度碎片化、含有RNA或化学污染物的DNA）都会影响文库制备。

有关如何对DNA样品进行质控，请参考起始DNA/RNA质控实验指南 。

化学污染物

从原始样本中提取DNA的方法不同，可能会导致经纯化的DNA中所残留的化学污染物不同。这会影响文库的制备效率和测序质量。请在牛津纳米孔社区的 Contaminants（污染物）页面 了解更多信息。

Custom primer mix sequences

Please order these sequences at 10 μM from IDT:

Forward primer: 5’ CAATTCGGTCTCCAGTGACTTGCCTGTCGCTCTATCTTC 3’ Reverse Primer: 5’ CAATTCGGTCTCCCACTTTTCTGTTGGTGCTGATATTGC 3’

Ligation Sequencing Kit (SQK-LSK110) contents

SQK-LSK110 kit contents

Name Acronym Cap colour No. of vials Fill volume per vial (µl)
DNA CS DCS Yellow 1 35
Adapter Mix F AMX-F Green 1 40
Ligation Buffer LNB Clear 1 200
L Fragment Buffer LFB White cap, orange stripe on label 2 1,800
S Fragment Buffer SFB Grey 2 1,800
Sequencing Buffer II SBII Red 1 500
Elution Buffer EB Black 1 200
Loading Beads II LBII Pink 1 360
Loading Solution LS White cap, pink sticker on label 1 360
Flush Buffer FB Blue 6 1,170
Flush Tether FLT Purple 1 200

3. 计算机要求及软件

MinION Mk1B的IT配置要求

请为MinION Mk1B配备一台高规格的计算机或笔记本电脑，以适配数据采集的速度。您可以在MinION Mk1B的IT配置要求文件中了解更多。

MinION Mk1C的IT配置要求

MinION Mk1C是一款集计算功能和触控屏幕于一体的便携式测序分析仪，它无需依赖任何额外设备，即可生成并分析纳米孔测序数据。您可以在 MinION Mk1C的IT配置要求文件中了解更多。

Software for nanopore sequencing

MinKNOW

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

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

EPI2ME (optional)

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

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

测序芯片质检

我们强烈建议您在开始测序实验前，对测序芯片的活性纳米孔数进行质检。质检需在您收到MinION /GridION /PremethION测序芯片12周之内进行，或者在您收到Flongle测序芯片四周内进行。Oxford Nanopore Technologies会对活性孔数量少于以下标准的芯片进行替换** ：

测序芯片 芯片上的活性孔数确保不少于
Flongle 测序芯片 50
MinION/GridION 测序芯片 800
PromethION 测序芯片 5000

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

4. DNA fragmentation

材料
  • 3 µg high molecular weight human genomic DNA
耗材
  • NEBNext dsDNA Fragmentase (M0348L)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
仪器
  • 涡旋混匀仪
  • Hula混匀仪（低速旋转式混匀仪）
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • 盛有冰的冰桶
  • 迷你离心机
  • 热循环仪
可选仪器
  • Agilent生物分析仪（或等效仪器）
  • Eppendorf 5424 离心机（或等效器材）

Prepare the DNA in nuclease-free water.

  • Transfer 3 μg genomic DNA into a 1.5 ml Eppendorf DNA LoBind tube
  • Adjust the volume to 16 μl with nuclease-free water
  • Mix thoroughly by flicking the tube to avoid unwanted shearing
  • Spin down briefly in a microfuge

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

Reagent Volume
DNA 16 µl
10x Fragmentase Reaction Buffer v2 2 µl
Total 18 µl

Vortex the tube for 3 seconds, and spin down.

Add 2 μl dsDNA Fragmentase to the tube.

Incubate the reaction for 28 minutes at 37°C.

提示

Fragmentation timing

Samples may fragment at different rates; 28 mins has shown to be the optimum time for NA12878 DNA extracted with the QIAGEN Genomic-tip. Users may wish to titrate fragmentation times of 25–32 mins if the fragmented yield is low.

Stop the fragmentation reaction by adding 5 μl of 0.5 M EDTA to the tube.

Vortex the tube for 3 seconds, and spin down.

Resuspend the AMPure XP beads by vortexing.

将样品转至干净的1.5 ml Eppendorf DNA LoBind 离心管中。

Add 15 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.

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

Spin down the sample and pellet the beads on a magnet for 5 mins.

Remove and retain the supernatant in a new 1.5 ml Eppendorf DNA LoBind tube.

Note: Do not discard the supernatant.

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

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

Prepare 500 μl of fresh 80% ethanol in nuclease-free water.

Spin down the sample and pellet the beads on a magnet for 5 minutes. Keep the tube on the magnet until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat the previous step.

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 pellet in 21 µl nuclease-free water. Incubate for 2 minutes at room temperature.

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 21 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Run a 1 μl aliquot on an Agilent Bioanalyzer to determine fragment length.

The fragment length distribution is expected to be similar to the trace below: Agilent

步骤结束

Take the fragmented DNA in 20 µl into the end-prep step. However, at this point it is also possible to store the sample at 4°C overnight.

5. End-prep

材料
  • Fragmented DNA in 20 µl
耗材
  • 0.2 ml薄壁PCR管
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 无核酸酶水（如ThermoFisher，AM9937）
  • NEBNext® Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • 新制备的80%乙醇（用无核酸酶水配制）
仪器
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P10 移液枪和枪头
  • 热循环仪
  • 迷你离心机
  • Hula混匀仪（低速旋转式混匀仪）
  • 磁力架
  • 盛有冰的冰桶

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

Between each addition, pipette mix 10-20 times.

Reagent Volume
Fragmented DNA 20 µl
Ultra II End-prep reaction buffer 7 µl
Ultra II End-prep enzyme mix 3 µl
Nuclease-free water 30 µl
Total 60 µl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Using a thermal cycler, incubate at 20°C for 30 minutes and 65°C for 30 mins.

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 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。

Prepare 500 μl of fresh 80% ethanol in nuclease-free water.

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。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol 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 pellet in 31 µl nuclease-free water. Incubate for 2 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 31 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of end-prepped DNA using a Qubit fluorometer.

步骤结束

Take forward ~300 ng of end-prepped DNA in 30 µl into adapter ligation. However, at this point it is also possible to store the sample at 4°C overnight.

6. Ligation of PCR adapters

材料
  • 300 ng end-prepped DNA in 30 µl
  • PCR Adapter (PCA)
耗材
  • 无核酸酶水（如ThermoFisher，AM9937）
  • NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • Hula混匀仪（低速旋转式混匀仪）
  • Qubit fluorometer (or equivalent for QC check)
  • 迷你离心机
  • 涡旋混匀仪

Add the reagents in the order given below, mixing by pipetting 10-20 times between each sequential addition:

Reagent Volume
End-prepped DNA 30 µl
PCR Adapters (PCA) 20 µl
NEB Blunt/TA Ligase Master Mix 50 µl
Total 100 µl

Ensure the components are thoroughly mixed by pipetting, and spin down.

室温下孵育10 分钟。

Resuspend the AMPure XP beads by vortexing.

Add 100 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.

End-prep cleanup demo

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。

Prepare 500 μl of fresh 80% ethanol in nuclease-free water.

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。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol 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 pellet in 49 µl nuclease-free water. Incubate for 2 minutes at room temperature.

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 49 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of adapted DNA using a Qubit fluorometer.

步骤结束

Take forward 48 µl of the adapted DNA into the PCR reaction. However, at this point it is also possible to store the sample at 4°C overnight.

7. PCR

材料
  • Primer Mix (PRM)
耗材
  • LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 0.2 ml 薄壁PCR管
仪器
  • 热循环仪
  • 盛有冰的冰桶
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • Hula混匀仪（低速旋转式混匀仪）
  • 涡旋混匀仪
  • Qubit fluorometer (or equivalent for QC check)

In a 0.2 ml thin-walled PCR tube mix the following:

Reagent Volume
LongAmp Taq 2X Master Mix 50 µl
PRM Adapters (10 μM) 2 µl
Template DNA 48 µl
Total 100 µl

Amplify using the following cycling conditions:

Cycle step Temperature Time No. of cycles
Initial denaturation 95 °C 3 mins 1
Denaturation 98 °C 20 secs 6 (b)
Annealing 62 °C (a) 15 secs (a) 6 (b)
Extension 65 °C (c) 3 mins 6 (b)
Final extension 65 °C 3 mins 1
Hold 4 °C ∞

a. This is specific to the Oxford Nanopore primer and should be maintained

b. Adjust accordingly if input quantities are altered

c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

Resuspend the AMPure XP beads by vortexing.

Add 100 μl of the resuspended AMPure XP beads to the sample, 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。

Prepare 500 μl of fresh 80% ethanol in nuclease-free water.

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。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol 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 35 µl nuclease-free water. Incubate for 2 minutes at room temperature.

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 2 µl of amplified DNA using a Qubit fluorometer.

步骤结束

Take forward 300–1000 ng amplified DNA into the hybridisation step. However, at this point it is also possible to store the sample at 4°C overnight.

8. Hybridisation

材料
  • Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
耗材
  • Cot-1 DNA (ThermoFisher Scientific 15279-011)
  • 无核酸酶水（如ThermoFisher，AM9937）
  • 0.2 ml薄壁PCR管
  • Blocking oligo at 1 mM, sequence 5'-AGGTTAAACACCCAAGCAGACGCCGCAATATCAGCACCAACAGAAACAACC-3'
仪器
  • SpeedVac
  • 热循环仪
  • 盛有冰的冰桶
  • 涡旋混匀仪
  • 迷你离心机

In a clean 1.5 ml Eppendorf DNA LoBind tube, mix the following:

Reagent Volume
DNA library 300–1000 ng
Cot-1 DNA 5 µg
Blocking oligo top 1 µl

The volume of the reaction can be variable, as the water is evaporated in step 2. After this, the DNA is reconstituted to a set volume.

Evaporate the water in a SpeedVac at 45ºC for approximately 1 hour.

Poke one or more holes in the lid with a narrow gauge needle. You can also break off the cap, cover with parafilm, and poke a hole in the parafilm.

Reconstitute with nuclease-free water to a final volume of 9 µl. Pipette up and down along the sides of the tube for optimal recovery.

Mix thoroughly by vortexing and spin down for 1 minute.

Move the 9 µl DNA library sample to a 0.2 µl thin-walled PCR tube, close the tube and incubate in the thermal cycler using the following program:

Stage Temperature Time
Step 1 95°C 5 min
Step 2 65°C 5 min
Step 3 65°C Hold

You will now need to prepare the Hybridisation Buffer mix and the RNase Block ready to be combined with the Capture Library reagent from the SureSelect kit. This will then be combined with the adapted, amplified DNA sample.

Once the sample tube is in the thermal cycler, mix the reagents in the table below to make the Hybridisation Buffer:

Reagent Volume for 1 reaction
SureSelect Hyb 1 (orange cap or bottle) 6.63 µl
SureSelect Hyb 2 (red cap) 0.27 µl
SureSelect Hyb 3 (yellow cap or bottle) 2.65 µl
SureSelect Hyb 4 (black cap or bottle) 3.45 µl
Total 13 µl

In the event of precipitation, warm the Hybridisation Buffer at 65°C for 5 minutes. Otherwise, keep buffer at room temperature until it is used for the Hybridisation mix.

Dilute the SureSelect RNase Block (purple cap) in nuclease-free water. Keep the mixture on ice.

RNase block dilution (parts RNase block:water) Volume of diluted RNase block
25% (1:3) 2 μl
重要

Please note that the 1:3 ratio of the RNase Block dilution is different to the SureSelect protocol.

Prepare the Capture Library Hybridisation Mix according to the table below. Only keep the mixture at room temperature until it is added to sample tube.

Reagent Volume for 1 reaction
Hybridisation buffer mixture 13 µl
25% RNase Block solution 2 µl
Capture library (red cap) ≥3 Mb 5 µl
Total 20 µl
重要

Do not keep solutions containing the Capture Library at room temperature for longer than 10 mins.

Keeping all reagents at 65°C, add 20 µl of the Capture Hybridisation Mix to the tube containing 9 µl adapted and amplified DNA sample.

吹打混匀。

Replace the cap on the tube.

重要

The tube must be closed to minimise evaporation and avoid a negative impact on your results.

Incubate the tube for 16–24 hours at 65°C with a heated lid set at 105°C.

步骤结束

Take your sample forward into the next step.

We do not recommend pausing your library prep at this stage by storing your sample at 4°C overnight.

9. Pull-down

材料
  • Sequence capture kit (e.g. Agilent SureSelect Human All Exon, Cat# 232866)
耗材
  • Dynabeads MyOne Streptavidin T1 (ThermoFisher Scientific, 65601)
  • 0.2 ml薄壁PCR管
  • 无核酸酶水（如ThermoFisher，AM9937）
仪器
  • 热循环仪
  • 涡旋混匀仪
  • 磁力架
  • 多通道移液枪和枪头
  • Plate mixer
  • 迷你离心机
  • 盛有冰的冰桶
重要

It is important to maintain bead suspensions at 65°C during the washing procedure below to ensure specificity of capture. Make sure that the SureSelect Wash Buffer 2 is pre-warmed to 65°C before use. Do not use a tissue incubator, or other devices with significant temperature fluctuations, for the incubation steps.

The hybrid capture protocol uses the SureSelect Target Enrichment Box 1 reagents (stored at room temperature), as well as Dynabeads MyOne Streptavidin T1 magnetic beads.

Warm the SureSelect Wash Buffer 2 at 65°C.

Resuspend the Dynabeads MyOne Streptavidin T1 magnetic beads by vortexing.

Add 50 µl of the beads to a fresh 1.5 ml Eppendorf DNA LoBind tube.

Add 200 µl of SureSelect Binding Buffer to the beads.

吹打混匀。

Place on a magnetic rack, allow beads to pellet and pipette off supernatant.

Repeat steps 4–6 twice more for a total of three washes.

Resuspend the beads in 200 µl of SureSelect Binding Buffer.

Keep the hybridisation tube at 65°C. Using a multichannel pipette, transfer the whole volume (~25–29 µl) of the hybridisation mixture from the 65°C reaction to the tube containing 200 µl of washed streptavidin beads.

Pipette up and down until beads are fully resuspended.

Incubate the tube on a Hula mixer for 30 mins at room temperature. Make sure the sample is mixing in the tube.

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。

Resuspend the beads in 200 µl of SureSelect Wash Buffer 1.

Pipette up and down until beads are fully resuspended.

Incubate the reaction for 15 minutes at room temperature.

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。

Resuspend the beads in 200 µl of Wash Buffer 2 pre-warmed at 65°C.

Pipette up and down until beads are fully resuspended.

Transfer the sample to a 0.2 ml thin-walled PCR tube.

Incubate the tube for 10 minutes at 65°C in the thermal cycler.

将样品转至干净的1.5 ml Eppendorf DNA LoBind 离心管中。

Place on a magnetic rack, allow beads to pellet and pipette off supernatant.

Repeat the wash steps 17–22 twice more for a total of three washes. Make sure all of the wash buffer has been removed during the final wash.

Add 96 µl of nuclease-free water to the sample, and pipette up and down to resuspend the beads. Keep the sample on ice.

Captured DNA remains on the streptavidin beads during the post-capture amplification step.

10. Elution and amplification of DNA

材料
  • Custom primer mix, 10 μM (IDT) - see below for sequences
耗材
  • 0.2 ml 薄壁PCR管
  • LongAmp Taq 2X Master Mix (e.g. NEB, cat # M0287)
  • 新制备的80%乙醇（用无核酸酶水配制）
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
  • 1.5 ml Eppendorf DNA LoBind离心管
仪器
  • 热循环仪
  • 盛有冰的冰桶
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • Hula混匀仪（低速旋转式混匀仪）
  • 涡旋混匀仪

Custom primer mix sequences

Please order these sequences at 10 μM from IDT:

Forward primer: 5’ CAATTCGGTCTCCAGTGACTTGCCTGTCGCTCTATCTTC 3’ Reverse Primer: 5’ CAATTCGGTCTCCCACTTTTCTGTTGGTGCTGATATTGC 3’

Split the sample into 2x 48 µl aliquots, and prepare the following reaction in duplicate.

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

Reagent Volume
LongAmp Taq 2x Master Mix 50 µl
Custom primer mix 2 µl
Template DNA 48 µl
Total 100 µl

Amplify using the following cycling conditions:

Cycle step Temperature Time No. of cycles
Initial denaturation 95°C 3 mins 1
Denaturation 98°C 20 secs 14 (b)
Annealing 62°C (a) 15 secs (a) 14 (b)
Extension 65°C (c) 3 mins 14 (b)
Final extension 65°C 3 mins 1
Hold 4°C ∞

a. This is specific to the primer mix and should be maintained

b. Adjust accordingly if input quantities are altered

c. This temperature is determined by the type of polymerase that is being used (given here for LongAmp Taq polymerase)

Place the amplified sample on a magnetic rack. Once the solution is clear, transfer the supernatant into a clean 1.5 ml Eppendorf DNA Lo-Bind tube. The beads can now be discarded.

Resuspend the AMPure XP beads by vortexing.

Add 180 µl of resuspended AMPure XP beads to the reaction and mix by pipetting.

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。

Prepare sufficient fresh 80% ethanol in nuclease-free water.

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。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol 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 pellet in 25 µl nuclease-free water. Incubate for 2 minutes at room temperature.

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 25 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Pool the two samples together to yield 50 µl eluted sample.

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

Run a 1 μl aliquot on an Agilent Bioanalyzer to determine fragment length.

The fragment length distribution is expected to be similar to the trace below: Agilent2

11. End-prep

材料
  • 500 ng–1 µg captured DNA in 48 µl
耗材
  • NEBNext Ultra II 末端修复/ dA尾添加模块（NEB，E7546）
  • 新制备的70%乙醇（用无核酸酶水配制）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • 无核酸酶水（如ThermoFisher，AM9937）
  • Agencourt AMPure XP beads (Beckman Coulter, A63881)
仪器
  • 热循环仪
  • 适用于1.5ml Eppendorf 离心管的磁力架
  • Hula混匀仪（低速旋转式混匀仪）
  • 涡旋混匀仪
  • 盛有冰的冰桶
可选仪器
  • Qubit fluorometer (or equivalent for QC check)

Perform end-repair and dA-tailing as follows:

Mix the following reagents in a 1.5 ml Eppendorf DNA LoBind tube:

Reagent Volume
DNA 48 µl
Ultra II End-prep reaction buffer 7 µl
Ultra II End-prep enzyme mix 3 µl
Nuclease-free water 2 µl
Total 60 µl

Mix gently by flicking the tube, and spin down.

Transfer the sample to a 0.2 ml thin-walled PCR tube.

Using a thermal cycler, incubate at 20°C for 30 minutes and 65°C for 30 mins.

Resuspend the AMPure XP beads by vortexing.

将样品转至干净的1.5 ml Eppendorf DNA LoBind 离心管中。

Add 60 µl of resuspended AMPure XP beads to the end-prep reaction and mix by pipetting.

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。

Prepare 500 μl of fresh 70% ethanol in nuclease-free water.

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。

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol 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 pellet in 31 µl nuclease-free water. Incubate for 2 minutes at room temperature.

将离心管静置于磁力架上，直到磁珠和液相分离，且洗脱液澄清无色。

Remove and retain 31 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of end-prepped DNA using a Qubit fluorometer - recovery aim >300 ng.

步骤结束

Take forward approximately 300 ng of end-prepped DNA in 30 µl into adapter ligation.

12. Adapter ligation and clean-up

材料
  • Adapter Mix F (AMX F)
  • 连接测序试剂盒内的连接缓冲液（LNB）
  • 短片段缓冲液（SFB）
  • Oxford Nanopore测序试剂盒中的洗脱缓冲液（EB）
耗材
  • NEBNext 快速连接模块（NEB，E6056）
  • 1.5 ml Eppendorf DNA LoBind 离心管
  • Agencourt AMPure XP beads (Beckman Coulter™, A63881)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
仪器
  • 磁力架
  • 迷你离心机
  • 涡旋混匀仪
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
  • P10 移液枪和枪头
重要

Although the recommended third-party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix (AMX) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.

Spin down the Adapter Mix F (AMX-F) and Quick T4 Ligase, and place on ice.

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

In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order:

Reagent Volume
DNA sample from the previous step 30 µl
Nuclease-free water 30 µl
Ligation Buffer (LNB) 25 µl
NEBNext Quick T4 DNA Ligase 10 µl
Adapter Mix F (AMX-F) 5 µl
Total 100 µl

Ensure the components are thoroughly mixed by pipetting, and spin down.

室温下孵育10分钟。

重要

If you have omitted the AMPure purification step after DNA repair and end-prep, do not incubate the reaction for longer than 10 minutes.

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) - do NOT use Long Fragment buffer (LFB). Flick the beads to resuspend, 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15µl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℃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15µl洗脱液转移至一支新的1.5ml Eppendorf DNA LoBind管中。

丢弃磁珠

CHECKPOINT

取1µl洗脱样品，用Qubit荧光计定量。

Use the average fragment size determined at the end of the “Elution and amplification of DNA” step to calculate the molarity of the sample.

Take 50 fmol of library and make up the volume to 12 μl with EB.

步骤结束

构建好的文库即可用于测序芯片上样。在上样前，请将文库置于冰上或4℃条件下保存。

提示

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.

可选操作

If quantities allow, the library may be diluted in Elution Buffer (EB) for splitting across multiple flow cells.

Additional buffer for doing this can be found in the Sequencing Auxiliary Vials expansion (EXP-AUX002), available to purchase separately. This expansion also contains additional vials of Sequencing Buffer II (SQII) and Loading Beads II (LBII), required for loading the libraries onto flow cells.

13. Priming and loading the SpotON flow cell

材料
  • Flush Buffer (FB)
  • Flush Tether (FLT)
  • Loading Beads II (LBII)
  • Sequencing Buffer II (SBII)
  • Loading Solution (LS)
耗材
  • 1.5 ml Eppendorf DNA LoBind 离心管
仪器
  • MinION device
  • SpotON Flow Cell
  • P1000 移液枪和枪头
  • P100 移液枪和枪头
  • P20 移液枪和枪头
提示

Priming and loading a MinION flow cell

We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.

Using the Loading Solution

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.

Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down at room temperature.

To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

Open the MinION device lid and slide the flow cell under the clip.

Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Flow Cell Loading Diagrams Step 1a

Flow Cell Loading Diagrams Step 1b

可选操作

为文库上样前，完成测序芯片检测，查看可用孔数目。

如此前已对测序芯片进行过质检，则此步骤可省略。

更多信息，请查看MinKNOW实验手册的 测序芯片质检 部分。

顺时针转动预处理孔孔盖，使预处理孔显露出来。

中文-测序芯片预处理上样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。

将预处理孔打开后，检查孔周围是否有小气泡。请按照以下方法，从孔中排出少量液体以清除气泡：

  1. 将P1000移液枪转至200µl刻度。
  2. 将枪头垂直插入预处理孔中。
  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。
    __请注意：__ 肉眼检查，确保从预处理孔到传感器阵列的缓冲液连续且无气泡。

中文-测序芯片预处理上样3

通过预处理孔向芯片中加入800µl预处理液，避免引入气泡。等待5分钟。在此期间，请按照以下步骤准备用于上样的DNA文库。

中文-测序芯片预处理上样4

Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.

重要

The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.

In a new tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer II (SBII) 37.5 µl
Loading Beads II (LBII) mixed immediately before use, or Loading Solution (LS), if using 25.5 µl
DNA library 12 µl
Total 75 µl

Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII).

完成测序芯片的预处理：

  1. 轻轻地翻起SpotON上样孔盖，使SpotON上样孔显露出来。 中文-测序芯片预处理上样5
  2. 通过预处理孔（而 非 SpotON加样孔）向芯片中加入200µl预处理液，避免引入气泡。 中文-测序芯片预处理上样6

临上样前，用移液枪轻轻吹打混匀制备好的文库。

通过SpotON加样孔向芯片中逐滴加入75µl样品。确保液滴流入孔内后，再加下一滴。

中文-测序芯片预处理上样7

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION device lid.

Flow Cell Loading Diagrams Step 8

Flow Cell Loading Diagrams Step 9

14. Data acquisition and basecalling

纳米孔数据分析概览

有关纳米孔数据分析的完整概述，包括碱基识别和次级分析，请参阅 数据分析 文档。

如何开始测序

MinKNOW软件负责仪器控制，数据采集和实时碱基识别。如您已在计算机上安装MinKNOW，则可选择以下几种途径开展测序：

1. 使用计算机上的MinKNOW进行实时数据采集和碱基识别

请按照 MinKNOW 实验指南 的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。

2. 使用GridION进行实时数据采集和碱基识别

请参照 GridION 用户手册 中的说明。

3. 使用MinION Mk1C测序仪进行实时数据采集和碱基识别

请参照 MinION Mk1C 使用指南中的说明。

4. 使用PromethION测序仪进行实时数据采集和碱基识别

请参照 PromethION 使用指南 或 PromethION 2 Solo 使用指南中的说明。

5. 使用计算机上的MinKNOW进行数据采集，过后再用NinKNOW进行线下碱基识别

请按照 MinKNOW 实验指南 中的说明：从“开始测序”部分起，到“MinKNOW运行结束”部分止。 当您设置实验参数时，请将 碱基识别 选项设为“关”。 测序实验结束后，请按照 MinKNOW 实验指南的本地分析 部分操作。

15. Downstream analysis

FASTQ Human Exome workflow in EPI2ME

Human Exome is an EPI2ME™ workflow for aligning basecalled reads to a set of reference sequences from Ensembl using the minimap2 aligner, covering the entire human exome. Alignments are performed against full gene sequences, including exons and introns, but currently not against flanking regions. The reference sequences are taken from the feature strand of the most recent human genome assembly, GRCh38.

The experiment report gives details on sequence length, accuracy, quality score and the amount of data generated during the experiment. It is possible to align to a number of human reference genes, and view more details about alignment coverage and accuracy.

For more information, please refer to the Workflows section in the EPI2ME protocol.

Suggested workflow

  1. Align reads to the human reference genome (GRCh38 or GRCh37) using minimap2. We recommend using -k parameter value 13
  2. (Optional) Remove reads that do not overlap with pull-down regions

Example data

Expected mean target coverage from one MinION Mk 1B flow cell: sequence depth

Aligned read length: Read length

Other data analysis options

1. Bioinformatics tutorials

For more in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials, which are available in the Bioinformatics resource section of the Community. The tutorials take the user through installing and running pre-built analysis pipelines, which generate a report with the results. The tutorials are aimed at biologists who would like to analyse data without the help of a dedicated bioinformatician, and who are comfortable using the command line.

2. Research analysis tools

Oxford Nanopore Technologies' Research division has created a number of analysis tools, which are available in the Oxford Nanopore GitHub repository. The tools are aimed at advanced users, and contain instructions for how to install and run the software. They are provided as-is, with minimal support.

3. Community-developed analysis tools

If a data analysis method for your research question is not provided in any of the resources above, we recommend the Community-developed data analysis tool library. Numerous members of the Nanopore Community have developed their own tools and pipelines for analysing nanopore sequencing data, most of which are available on GitHub. Please be aware that these tools are not supported by Oxford Nanopore Technologies, and are not guaranteed to be compatible with the latest chemistry/software configuration.

16. 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。

17. 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。

18. Issues during the sequencing run

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。

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol"
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the sequencing kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FLT/FCT tube). Make sure FLT/FCT was added to FB/FCF before priming.

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。

运行过程中过孔速度和数据质量（Q值）降低

现象 可能原因 措施及备注
运行过程中过孔速度和数据质量（Q值）降低 对试剂盒9系列试剂（如SQK-LSK109），当测序芯片的上样量过多时（请参阅相应实验指南获取推荐文库用量），能量消耗通常会加快。 请按照MinKNOW 实验指南中的说明为测序芯片补充能量。请在后续实验中减少测序芯片的上样量。

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。

Guppy – no input .fast5 was found or basecalled

Observation Possible cause Comments and actions
No input .fast5 was found or basecalled input_path did not point to the .fast5 file location The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH.
No input .fast5 was found or basecalled The .fast5 files were in a subfolder at the input_path location To allow Guppy to look into subfolders, add the --recursive flag to the command

Guppy – no Pass or Fail folders were generated after basecalling

Observation Possible cause Comments and actions
No Pass or Fail folders were generated after basecalling The --qscore_filtering flag was not included in the command The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders.

Guppy – unusually slow processing on a GPU computer

Observation Possible cause Comments and actions
Unusually slow processing on a GPU computer The --device flag wasn't included in the command The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command.

Last updated: 3/10/2023

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