Ligation sequencing gDNA V14 — human sample (N50 10 kb) on PromethION (SQK-LSK114)
PromethION: Protocol
Ligation sequencing gDNA V14 — human sample (N50 10 kb) on PromethION (SQK-LSK114) V GDH_9173_v114_revP_10Nov2022
- This protocol uses N50 of 10 kb genomic DNA extracted from human cell lines
- Sample preparation time: ~220 minutes and library preparation time: ~60 minutes
- Data analysis: 1-2 hours
- No PCR
- Compatible with R10.4.1 flow cells
For Research Use Only
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Sample preparation
- 3. Human cell line DNA extraction using the QIAGEN Puregene Cell Kit
- 4. Shearing DNA for 10 kb input using the Covaris g-TUBE™
Library preparation
Sequencing and data analysis
Troubleshooting
概览
- This protocol uses N50 of 10 kb genomic DNA extracted from human cell lines
- Sample preparation time: ~220 minutes and library preparation time: ~60 minutes
- Data analysis: 1-2 hours
- No PCR
- Compatible with R10.4.1 flow cells
For Research Use Only
1. Overview of the protocol
Introduction to the protocol for sequencing 10 kb human DNA on PromethION using the Ligation Sequencing Kit V14 (SQK-LSK114)
This protocol describes an end-to-end process to prepare, sequence gDNA from cultured cells samples and analyse using the “Human variant workflow” in Epi2Me. The identification of structural variants (SVs) and single nucleotide variants (SNVs) play a pivotal role in our understanding of genetic diversity, disease mechanisms, and evolutionary biology1,2. The protocol aims to produce libraries with a read N50 ~10 kb and generate ~30-40x coverage of the genome, to provide robust calling of small and large variants as well as provide methylation and phasing information.
Briefly, genomic DNA is extracted from 5 million cultured cells, and is extracted using the QIAGEN Puregene Cell Kit. DNA is then sheared with the Covaris g-TUBE™, and libraries prepared with the Ligation Sequencing Kit V14 (SQK-LSK114). Libraries are then sequenced on PromethION.
Data is basecalled and aligned by MinKNOW and the aligned BAM output data is analysed using the wf-human-variation workflow which uses Sniffles2, Clair3 and modbam2bed software to call structural variants (SVs), single nucleotide polymorphisms (SNPs) and for reporting DNA methylation.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your input sample (cells)
- 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
Sample preparation
Using the outlined extraction method, extract the gDNA from your cells, and fragment your gDNA using the Covaris g-TUBE.
Check the length, quantity and purity of your extracted material. The quality checks performed during the protocol are essential in ensuring experimental success.
Library preparation
The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.
| Library preparation | Process | Time | Stop option |
|---|---|---|---|
| DNA repair and end-prep | Repair and prepare the DNA ends for adapter attachment | 35 minutes | 4°C overnight |
| Adapter ligation and clean-up | Attach the sequencing adapters to the DNA ends | 20 minutes | 4°C short-term storage or for repeated use, such as re-loading your flow cell -80°C for single-use, long-term storage. We strongly recommend sequencing your library as soon as it is adapted. |
| Priming and loading the flow cell | Prime the flow cell and load the prepared library for sequencing | 5 minutes |
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.
- Analyse data using the wf-human-variation workflow.
重要
实验方案适用性 (2)
本实验方案仅适用于与以下产品搭配使用：
- 连接测序试剂盒V14（SQK-LSK114）
- Lambda标准品扩展包（EXP-CTL001）
- R10.4.1 PromethION 测序芯片 (FLO-PRO114M)
- 测序芯片清洗剂盒（EXP-WSH004）
- PromethION 24/48 测序设备 - PromethION IT 配置要求文件
- PromethION 2 Solo 测序设备 - PromethION 2 Solo IT 配置要求文件
2. Equipment and consumables
材料
- (FOR EXTRACTION) 5 x 10⁶ cells (e.g. cell culture or tissue sample)
- (FOR LIBRARY PREPARATION) 1 µg of fragmented gDNA
- 连接测序试剂盒V14（SQK-LSK114）
耗材
- PromethION 测序芯片
- 供Oxford Nanopore Technologies®连接测序使用的NEBNext®配套模块v2（NEB, E7672S 或 E7672L）
- Puregene Cell Kit (QIAGEN, 158043)
- g-TUBE™ (Covaris, 520079)
- 15 ml Falcon tubes
- 2 ml Eppendorf DNA LoBind 离心管
- 1.5 ml Eppendorf DNA LoBind 离心管
- 0.2 ml 薄壁PCR管
- 无核酸酶水（如ThermoFisher，AM9937）
- 新制备的80%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)
- (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
- Inoculation loop or disposable tweezers
- Qubit™ 分析管（Invitrogen, Q32856）
- 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）
- Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
仪器
- PromethION测序设备
- PromethION 测序芯片遮光片
- 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
- Incubator or water bath set at 37°C and 50°C
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- 迷你离心机
- 涡旋混匀仪
- 热循环仪
- Wide-bore pipette tips
- 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
- 盛有冰的冰桶
- 计时器
- Agilent Femto Pulse System (or equivalent for read length QC)
- Qubit荧光计（或用于质控检测的等效仪器）
重要
The above list of materials, consumables, and equipment is for the extraction method in the sample preparation section, as well as the library preparation section of the protocol. If you have pre-extracted sample(s), you will only require the materials for the library preparation section of this protocol.
For the library preparation protocol, you will need 3 µg of genomic DNA with an N50 of 10 kb.
For this end-to-end workflow, we recommend extracting high molecular weight human gDNA from from 5 x 10⁶ cells (e.g. cell culture or tissue sample) using the QIAGEN Puregene Cell Kit in the Sample Preparation step.
Other extraction protocols are available but have not been tested by Oxford Nanopore Technologies.
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。
提示
Eppendorf tube orientation in centrifuge
For all centrifugation steps, ensure that tubes are loaded into the centrifuge with the hinge side of the tube facing outwards. This will assist in visual identification of the pellet.
Ensure gentle handling when removing the tubes from the centrifuge to avoid dislodging the pellet.
提示
我们建议您使用专供Oxford Nanopore Technologies®连接测序的NEBNext® 配套模块v2（目录号E7672S或E7672L）。该配套模块内包含所有与连接测序试剂盒配套使用的NEB试剂。
之前版本的NEBNext® 配套模块（NEB，E7180S或E7180L）虽然兼容，但v2版在dA尾添加和连接步骤上的效率更高。
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.
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。
重要
为确保高效接头（LA）连接，我们强烈建议您使用连接测序试剂盒V14中提供的连接缓冲液（LNB）而非其它第三方连接酶缓冲液。
重要
本试剂盒所用连接接头（LA）经过升级，不可与其它测序接头互换使用。
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： 
部分试剂改为瓶装： 
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。
3. Human cell line DNA extraction using the QIAGEN Puregene Cell Kit
材料
- 5 x 10⁶ cells (e.g. cell culture or tissue sample)
耗材
- Puregene Cell Kit (QIAGEN, 158043)
- Freshly prepared cold 70% ethanol in nuclease-free water
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- 15 ml Falcon tubes
- 2 ml Eppendorf DNA LoBind 离心管
- 1.5 ml Eppendorf DNA LoBind 离心管
- Inoculation loop or disposable tweezers
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ 分析管（Invitrogen, Q32856）
仪器
- Incubator or water bath set at 37°C and 50°C
- Centrifuge and rotor suitable for 15 ml Falcon tubes
- Vortex mixer
- Wide-bore pipette tips
- P1000移液枪和枪头
- P200 移液枪和枪头
- 盛有冰的冰桶
- Qubit荧光计（或用于质控检测的等效仪器）
Prepare a 1.5 ml Eppendorf DNA LoBind tube with approximately 1 ml of 70% ethanol and store on ice to cool.
We recommend to harvest and pellet 5 x 10⁶ cells in a 1.5 ml Eppendorf DNA LoBind tube. However, this may vary depending on your sample type. If any liquid remains associated with the pellet, spin down the cells again, then aspirate and discard the remaining supernatant.
Add 200 µl of 1x PBS to pelleted cells.
Centrifuge at 300 x g for 3 minutes.
Without disturbing the pellet, aspirate and discard the supernatant.
Add 3 ml of Cell Lysis Solution, and pipet up and down gently to lyse the cells and homogenise the solution until no clumps remain.
Ensure that the solution is homogenous.
Transfer the resuspended cells to a 15 ml Falcon. If clumps of cells remain, gently invert the tube to ensure resuspension.
Incubate the sample at 37°C for 30 minutes.
Add 1000 µl of the Protein Precipitation Solution to the lysed cells and mix by vortexing for three pulses of 5 seconds.
Centrifuge the sample at 2000 x g for 5 minutes.
The precipitated protein should form a tight, reddish-brown pellet.
If the protein pellet is not tight, incubate on ice for 5 minutes and repeat the centrifugation.
Add 3 ml of isopropanol into a clean 15 ml falcon tube.
Mix by gently inverting the tube 50 times until the DNA is visible as threads or a clump.
Spool the DNA using an inoculation loop or disposable tweezers.
Briefly dip the spooled DNA in the 1.5 ml Eppendorf DNA LoBind tube containing cold 80% ethanol and allow to air dry for a few seconds.
Transfer the inoculation loop or tweezers with the spooled DNA to a 1.5 ml Eppendorf DNA LoBind tube containing 250 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and allow the DNA to gently dislodge from the loop/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.
Note: The pellet may take some time to dissolve. Ensure the solution is homogenous before proceeding to quantification by gentle inversion and spin down.
Quantify the sample three times using the Qubit dsDNA BR Assay Kit, ensuring that replicate Qubit measurements are consistent before continuing to the next step.
If the Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended. If this occurs, we recommend increasing the incubation time to aid with resuspension of the DNA pellet.
步骤结束
Take the sample in 250 µl TE buffer forwards into the next step or the sample can be stored at 4°C overnight.
4. Shearing DNA for 10 kb input using the Covaris g-TUBE™
材料
- 2 µg of extracted high molecular weight gDNA
耗材
- g-TUBE™ (Covaris, 520079)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- 1.5 ml Eppendorf DNA LoBind 离心管
- (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ 分析管（Invitrogen, Q32856）
- Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
仪器
- 迷你离心机
- P200 移液枪和枪头
- P100 pipette and tips
- P20 移液枪和枪头
- P2 移液枪和枪头
- Agilent Femto Pulse System (or equivalent for read length QC)
- Qubit荧光计（或用于质控检测的等效仪器）
可选操作
We recommend using TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) rather than nuclease-free water if the library is going to be stored over a long-term period.
Transfer 2 μg extracted gDNA into a 1.5 ml Eppendorf DNA LoBind tube, and adjust the volume to 100 μl with nuclease-free water.
Mix the DNA thoroughly by flicking the tube. Spin down briefly in a microfuge.
Transfer the genomic DNA sample in 100 μl to a Covaris g-TUBE™.
For a fragment length of 10 kb, centrifuge the g-TUBE™ at 4300 x g for one minute at room temperature. Remove and check that all the DNA has passed through the tube.
Note: Please ensure that you are using the correct speed for your equipment and input sample to achieve shearing of 10 kb fragment lengths.
提示
If DNA remains in the upper chamber of the Covaris g-TUBE™, spin again for one minute at the same speed.
Invert the g-TUBE™ and centrifuge again for one minute to collect the fragmented DNA. Remove and check that all the DNA has passed through the tube.
提示
If DNA remains in the upper chamber of the Covaris g-TUBE™, spin again for one minute at the same speed.
Transfer the 100 μl fragmented DNA into a clean 1.5 ml Eppendorf DNA LoBind tube.
CHECKPOINT
Quantify 1 µl of eluted sample using a Qubit fluorometer and check sample read length using the Agilent Femto Pulse System. An N50 of 10 kb read lengths is to be expected.
步骤结束
Take forwards 1 μg sample into the next step or the sample can be stored at 4°C overnight.
5. DNA损伤及末端修复 (3)
材料
- 1 µg of fragmented gDNA
- AMPure XP 磁珠（AXP）
耗材
- NEBNext®配套模块v2（NEB，E7672S或E7672L）中的NEBNext® FFPE DNA修复混合液（NEB，M6630）
- NEBNext®配套模块v2（NEB，E7672S或E7672L）中的NEBNext® FFPE DNA修复缓冲液v2（E7363）
- NEBNext®配套模块v2（NEB，E7672S或E7672L）中的NEBNext® Ultra II 末端修复酶混合物（E7646）
- Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
- 无核酸酶水（如ThermoFisher，AM9937）
- 新制备的80%乙醇（用无核酸酶水配制）
- Qubit™ 分析管（Invitrogen, Q32856）
- 0.2 ml薄壁PCR管
- 1.5 ml Eppendorf DNA LoBind 离心管
仪器
- P1000 移液枪和枪头
- P100 移液枪和枪头
- P10 移液枪和枪头
- 迷你离心机
- 热循环仪
- Hula混匀仪（低速旋转式混匀仪）
- 磁力架
- 盛有冰的冰桶
可选仪器
- Qubit荧光计（或用于质控检测的等效仪器）
根据生产厂家的说明准备NEB试剂，并置于冰上。
为获得最优表现，NEB建议如下：
于冰上解冻所有试剂。
轻弹并/或翻转各管，确保各试剂充分混匀。
注意： 请切勿涡旋振荡 FFPE DNA修复混合液或 Ultra II末端修复酶混合物。同一日内首次打开一管试剂前，请务必先将该管试剂瞬时离心。
涡旋振荡 FFPE DNA 修复缓冲液 v2或FFPE DNA 修复缓冲液、及 Ultra II 末端修复反应缓冲液，确保混匀。
注意： 上述缓冲液中可能会出现白色沉淀。如发现沉淀，请待液体回复至室温后，使用移液枪上下吹打数次，打散沉淀；然后快速涡旋振荡混匀。FFPE DNA 修复缓冲液可能轻微泛黄，不影响使用。
用无核酸酶水稀释DNA： (3)
将1μg（或100-200 fmol）基因组DNA转移至一支1.5ml Eppendorf DNA LoBind离心管中。
如不足47 μl，请加入无核酸酶水补足。
用移液枪吹打离心管或轻弹离心管以充分混匀。
使用迷你离心机快速离心。
在一支0.2ml的薄壁PCR管中，混合以下试剂： (3)
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 the reaction at 20°C for 5 minutes, then 65°C for 5 minutes and hold at 4°C.
涡旋振荡以重悬AMPure XP磁珠（AXP）。
将DNA样本转至干净的1.5 ml Eppendorf DNA LoBind离心管中。
将60µl重悬的AMPure XP磁珠（AXP）加入DNA末端修复反应体系中，轻弹试管以充分混合。
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 600 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。 (1)
Keep the tube on the magnet and wash the beads with 250 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。
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。 (3)
步骤结束
经过末端修复的DNA可用于稍后的接头连接。如需要，您也可以此时将样品置于4℃储存过夜。 (2)
6. Adapter ligation and clean-up
材料
- 连接接头（LA）
- 连接测序试剂盒内的连接缓冲液（LNB）
- 长片段缓冲液（LFB）
- AMPure XP 磁珠（AXP）
- Elution Buffer (EB) from the Ligation Sequencing Kit
耗材
- Salt-T4® DNA Ligase (NEB, M0467)
- 1.5 ml Eppendorf DNA LoBind 离心管
- Qubit™ 分析管（Invitrogen, Q32856）
- Qubit dsDNA HS Assay（双链DNA高灵敏度检测）试剂盒（Invitrogen, Q32851）
仪器
- Hula混匀仪（低速旋转式混匀仪）
- 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
- Qubit荧光计（或用于质控检测的等效仪器）
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）和耐盐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 Long Fragment Buffer (LFB) 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：
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。
涡旋振荡以重悬AMPure XP磁珠（AXP）。
将40µl 重悬的AMPure XP磁珠加入反应体系中，轻弹离心管以充分混合。
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 Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet for at least 5 minutes. Remove the supernatant using a pipette and discard.
Note: Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.
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。
Remove the tube from the magnetic rack and resuspend the pellet in 33 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at 37°C.
Pellet the beads on a magnet for 10 minutes until the eluate is clear and colourless.
Remove and retain 33 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Dispose of the pelleted beads
CHECKPOINT
取1µl洗脱样品，用Qubit荧光计定量。
重要
For libraries with an N50 of 10 kb, we recommend loading at least 200-300 ng (35-50 fmol) of your final prepared library onto the R10.4.1 flow cell.
This protocol has been optimised for output to achieve the analysis of one genome at 30X coverage.
It is vital to ensure 200-300 ng of 10 kb input is loaded onto the flow cell to maximise pore occupancy from the beginning of the run for optimal sequencing. If pore occupancy is low at the beginning of a run, more terminal blocking will occur throughout sequencing which will result in lower outputs.
If required, we recommend using a mass to mol calculator such as the NEB calculator.
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
文库保存建议
若为 短期 保存或重复使用（例如在清洗芯片后再次上样），我们建议将文库置于Eppendorf LoBind 离心管中 4℃ 保存。 若为一次性使用且储存时长 __超过3个月__，我们建议将文库置于Eppendorf LoBind 离心管中 -80℃ 保存。
7. PromethION 测序芯片的预处理及上样
材料
- 测序缓冲液（SB）
- 文库颗粒（LIB）
- 文库溶液（LIS）
- 测序芯片系绳（FCT）
- 测序芯片冲洗液（FCF）
耗材
- PromethION 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
本试剂盒仅兼容R10.4.1测序芯片（FLO-PRO114M）。
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。
按下表制备测序芯片的预处理液，室温下涡旋振荡混匀。
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。请按照与您所用试剂盒包装相对应的说明操作。
单次使用管装： 向一整管测序芯片冲洗液（FCF）中加入30µl 测序芯片系绳（FCT）。
瓶装： 请另拿一支适当体积的离心管制备测序芯片预处理液：
| 试剂 | 体积（每张芯片） |
|---|---|
| 测序芯片冲洗液 (FCF) | 1,170 µl |
| 测序芯片系绳 (FCT) | 30 µl |
| 总体积 | 1,200 µl |
重要
将芯片从冰箱中取出后，请将其置于室温环境孵育20分钟再插入PromethION测序仪。潮湿环境下的测序芯片上可能会形成冷凝水。因此，请检查测序芯片顶部和底部的金色连接器引脚处是否有水凝结。如有，请使用无纤维布擦干。请确保测序芯片底部有热垫（黑色）覆盖。
对 PromethION 2 Solo，请按以下步骤为测序芯片上样：
将测序芯片平放在金属板上。
将测序芯片推入对接端口，直至金色引脚或绿色电路板不可见。
对PromethION 24/48，将测序芯片插入相应卡槽的对接端口：
将测序芯片与连接器横竖对齐，以便顺利卡入。
用力下压芯片至卡槽，并确认卡夹位置归位。
重要
如插入配置测试芯片的角度出现偏差，可能会损坏PromethION上的引脚并影响测序结果。如您发现 PromethION测序仪芯片位置上的引脚损坏，请通过电子邮件（support@nanoporetech.com）或微信公众号在线支持（NanoporeSupport）联系我们的技术支持团队。
顺时针滑动加液孔孔盖，将其打开。
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：
- 将P1000移液枪转至200µl刻度。
- 将枪头垂直插入加液孔中。
- 反向转动移液枪量程调节转纽，直至移液枪刻度在220-230 µl之间，或直至您看到有少量缓冲液进入移液枪枪头。
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。
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：
| 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。
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。
合上加液孔孔盖。
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：
- 将遮光片的中空部分（空槽）与测序芯片的加液孔孔盖对齐。确保遮光片的前沿位于测序芯片ID的上方。
- 用力下压遮光片的卡垫部分，遮光片空槽边缘会随卡垫卡入加液孔孔盖下方。
步骤结束
准备就绪后，合上PromethION设备上盖。
请在为PromethION芯片上样后，等待10分钟再启动实验，以提高芯片产出。
8. Data acquisition and basecalling
How to start sequencing
The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software.
We recommend basecalling with the high accuracy (HAC) basecaller in real-time with BAM selected as output type using the P2 Solo or P24/P48 device.
You must generate a BAM file from your sequening run, as this is required for input into the wf-human-variation workflow.
Refer to the links below containing the detailed instructions for setting up the device and sequencing run:
PromethION 24 and 48: "Starting a sequencing run with PromethION 24 and 48"
PromethION 2 Solo: "Starting a sequencing run on PromethION 2 Solo"
Below are the recommended sequencing parameters for MinKNOW.
MinKNOW settings for 10kb human sample on PromethION
We recommend using the modified bases option for basecalling and ensuring a BAM output is selected when setting up your MinKNOW run. The remaining sequencing parameters are kept to default. Below are our current recommendations:
Positions
Flow cell position: [user defined]
Experiment name: [user defined]
Flow cell type: FLO-PRO114M
Sample ID: [user defined]
Kit
Kit selection: Ligation Sequencing Kit (SQK-LSK114)
Run configuration
Sequencing and analysis
Basecalling: On [default] Modified bases: On with '5mC & 5hmC CG contexts' selected Model: High-accuracy basecalling (HAC) [default]
Barcoding: Disabled [default]
Alignment: Off [default]
We do not currently recommend live alignment during sequencing, as it can slow down system processing.
Adaptive sampling: Off [default]
Advanced options Active channel selection: On [default] Time between pore scans: 1.5 [default] Reserve pores: On [default]
Data targets
Run limit: 72 hours [default]
Output
Output format .POD5: On [default] .FASTQ: On [default] .BAM: On
Filtering: On [default] Qscore: 9 [default] Minimum read length: 200 bp [default]
重要
We do not recommend live alignment during sequencing, as it can slow down system processing.
You can align your BAM file post-sequencing by following one of the methods below:
| Aligning the BAM file in MinKNOW | Aligning the BAM file during the wf-human-variation workflow |
|---|---|
| Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing. The aligned BAM file can be used as your file input in the wf-human-variation workflow. Using mapped BAM as input, the workflow will take 1-2 hours. | You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up. Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours. |
Further information is available in the Downstream analysis section of this protocol.
9. Downstream analysis
Analysis of human cell DNA sequence data
For the analysis of human cell DNA sequence data, we recommend the wf-human-variation workflow. This end-to-end software pipeline is implemented using the Nextflow workflow language and implements methods for the calling of single nucleotide polymorphisms (SNPs), structural variants (SVs), and for reporting DNA methylation information.
The wf-human-variation workflow is best run from the BAM file produced by MinKNOW when the modified base model for basecalling is selected. If sequence read mapping to the reference genome is not performed by MinKNOW, we recommend to perform the basecalling using the wf-basecalling workflow. Ensure you save the outputs in BAM format by providing the --output_bam option.
The tools below are used in the analysis workflow and can be run in isolation or together:
Sniffles2 calls SVs and file output include an HTML report of QC metrics and VCF format list of variants and their quality scores.
Clair3 calls SNPs and file output includes an HTML report of QC metrics and VCF format list of variants and their quality scores.
modkit extracts methylation information from the provided BAM file which is summarised in a BEDmethyl format file.
The wf-human-variation workflow is preconfigured using appropriate parameters and requires tuning only for the choice of reference genome and Clair3 model. Please see the project’s documentation for further details.
The results from the wf-human-variation workflow can be further explored by viewing in a track-based genome browser such as IGV can be assessed for known pathogenicity through tertiary analysis software.
EPI2ME analysis workflow
The wf-human-variation workflow is intended to be run using the Nextflow software.
We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a Graphical User Interfaces (GUI). EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis. The collection of all our available EPI2ME workflows can be found here.
For new users, the quick start guide can be found here outlining how to use this interface.
重要
Compute requirements for the wf-human-variation workflow on EPI2ME
| Recommended requirements | Minimum requirements |
|---|---|
| CPUs = 32 | CPUs = 16 |
| Memory = 128GB | Memory = 32GB |
Approximate run time: Variable depending on whether it is targeted sequencing or whole genome sequencing, as well as coverage and the individual analyses requested. For instance, a 90X human sample run (options: --snp --sv --mod --str --cnv --phased --sex male) takes less than 8h with recommended resources.
ARM processor support: False
可选操作
The wf-human-variation workflow can also be run using the command line interface (CLI)
Please see the Github page for further details.
Note: We only recommend the command line interface (CLI) for experienced users.
Open the EPI2ME app using the desktop shortcut.
On the landing page, open the workflow tab on the left-hand sidebar.
Navigate to the Available workflows tab and click on wf-human-variation option.
Click install.
Navigate to the Installed tab and click on the installed wf-human-variation workflow.
可选操作
If the workflow was already installed, check for updates by clicking 'Update workflow'.
We recommend running the latest version of our workflows for the best results.
Click on Run this workflow to open the launch wizard.
Select the environment you are running the workflow from:
Click on the sub-workflow(s) you want to run in the wf-human-variation analysis.
You must have at least one of the sub-workflows selected to proceed with analysis.
Note: For more information on the sub-workflows click on the "Expand" option in the platform, or visit our online EPI2ME documentation.
Navigate to the 'Main options' tab to assign a 'Sample name' as an identifier in workflow outputs.
重要
The wf-human-variation workflow uses sequencing data in the form of a single BAM file or a folder of BAM files.
The BAM files used as an input can be aligned or unaligned:
| Aligning the BAM file in MinKNOW (prior to the wf-human-variation workflow) | Aligning the BAM file during the wf-human-variation workflow (during the wf-human-variation workflow) |
|---|---|
| Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing. The aligned BAM file can be used as your file input in the wf-human-variation workflow. For more information on post-run alignment in MinKNOW please visit our MinKNOW protocol. Using mapped BAM as input, the workflow will take 1-2 hours. | You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up. Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours. |
In the 'Main options' upload your sequencing data in the form of a single BAM file or a folder of BAM files.
可选操作
If you have an unaligned BAM file as input, in the 'Main options' upload your reference genome in FASTA format.
Click Launch workflow.
Ensure all parameter options have green ticks.
Once the wf-human-variation workflow finishes, a report will be produced alongside output files.
wf-human-variation workflow outputs
The primary workflow outputs include:
- gzipped VCF file containing the SNPs in the dataset from
--snp - gzipped VCF file containing the SVs in the dataset from
--sv - gzipped bedMethyl file aggregating modified base counts from
--mod - HTML report detailing the primary findings of the workflow for QC metrics, and SNP and SV calling
- If an unaligned BAM file was provided, the workflow will ouput a CRAM file containing the alignments used to make the downstream variant calls.
The secondary workflow outputs:
mosdepthouputs include:{sample_name}.mosdepth.global.dist.txt: a cumulative distribution indicating the proportion of total bases for each and all reference sequences{sample_name}.regions.bed.gz: the mean coverage for each region in the provided BED file{sample_name}.thresholds.bed.gz: the number of bases in each region that are covered at or above each threshold value (1, 10, 20, 30X)
- bamstats ouputs include:
{sample_name}.readstats.tsv.gz: a gzipped TSV summarising per-alignment statistics produced by bamstats{sample_name}.ftagstat.tsv: a text file with summary alignment statistics for each reference sequence
wf-human-variation workflow tips
It is possible to phase SNPs, SVs and modified bases by providing the --phased option.
To improve the accuracy of SV calling, specify a suitable tandem repeat BED for your reference with --tr_bed.
Aggregation of methylation calls with --mod requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags. To do so on MinKNOW, ensure 'Modified bases' option is selected during basecalling set up, with the '5mC' model selected.
Ensure to retain the input reference when basecalling or alignment is performed as CRAM files cannot be read without the corresponding input reference.
For a full list of available basecalling models, refer to the Dorado documentation.
10. 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至+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。
或者，请按照回收程序将测序芯片返还至Oxford Nanopore。
您可在此处找到回收测序芯片的说明。
重要
如果您遇到问题或对测序实验有疑问，请参阅本实验指南在线版本中的“疑难解答指南”一节。
11. Issues during DNA extraction and library preparation
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。
Low sample quality
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2) | The DNA extraction method does not provide the required purity | The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional AMPure bead clean-up step. |
经AMPure磁珠纯化后的DNA回收率低
| 现象 | 可能原因 | 措施及备注 |
|---|---|---|
| 低回收率 | AMPure磁珠量与样品量的比例低于预期，导致DNA因未被捕获而丢失 | 1. AMPure磁珠的沉降速度很快。因此临加入磁珠至样品前，请确保将磁珠重悬充分混匀。 2. 当AMPure磁珠量与样品量的比值低于0.4:1时，所有的DNA片段都会在纯化过程中丢失。 |
| 低回收率 | DNA片段短于预期 | AMPure磁珠量与样品量的比值越低，针对短片段的筛选就越严格。每次实验时，请先使用琼脂糖凝胶（或其他凝胶电泳方法）确定起始DNA的长度，并据此计算出合适的AMPure磁珠用量。  |
| 末端修复后的DNA回收率低 | 清洗步骤所用乙醇的浓度低于70% | 当乙醇浓度低于70%时，DNA会从磁珠上洗脱下来。请确保使用正确浓度的乙醇。 |
12. 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 | For the human genome sequencing protocols, 200-300 ng of good quality library should be loaded on to an R10.4.1 flow cell to keep pore occupancy high. |
| 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 SQK-LSK114 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 (FCT tube). Make sure FCT was added to 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。  在上图中，样本1为高分子量DNA，而样本2为降解样本。3. 在制备文库的过程中，请避免使用吹打或/和涡旋振荡的方式来混合试剂。轻弹或上下颠倒离心管即可。 |
大量纳米孔处于不可用状态
| 现象 | 可能原因 | Comments and actions |
|---|---|---|
大量纳米孔处于不可用状态 (在通道面板和纳米孔活动状态图上以蓝色表示)  上方的纳米孔活动状态图显示：状态为不可用的纳米孔的比例随着测序进程而不断增加。 | 样本中含有污染物 | 使用MinKNOW中的“Unblocking”（疏通）功能，可对一些污染物进行清除。 如疏通成功，纳米孔的状态会变为"测序孔". 若疏通后，状态为不可用的纳米孔的比例仍然很高甚至增加： 1. 用户可使用 测序芯片冲洗试剂盒（EXP-WSH004）进行核酸酶冲洗 can be performed, 操作，或 2. 使用PCR扩增目标片段，以稀释可能导致问题的污染物。 |
大量纳米孔处于失活状态 (1)
| 现象 | 可能原因 | 措施及备注 |
|---|---|---|
| 大量纳米孔处于失活状态（在通道面板和纳米孔活动状态图上以浅蓝色表示。膜结构或纳米孔遭受不可逆转地损伤） | 测序芯片中引入了气泡 | 在芯片预处理和文库上样过程中引入的气泡会对纳米孔带来不可逆转地损害。请观看 测序芯片的预处理及上样 视频了解最佳操作方法。 |
| 大量纳米孔处于失活/不可用状态 | 文库中存在与DNA共纯化的化合物 | 与植物基因组DNA相关的多糖通常能与DNA一同纯化出来。 1. 请参考 植物叶片DNA提取方法。 2. 使用QIAGEN PowerClean Pro试剂盒进行纯化。 3. 利用QIAGEN REPLI-g试剂盒对原始gDNA样本进行全基因组扩增。 |
| 大量纳米孔处于失活/不可用状态 | 样本中含有污染物 | 您可在 污染物专题技术文档 中查看污染物对测序实验的影响。请尝试其它不会导致污染物残留的提取方法。 |
温度波动
| 现象 | 可能原因 | 措施及备注 |
|---|---|---|
| 温度波动 | 测序芯片和仪器接触不良 | 检查芯片背面的金属板是否有热垫覆盖。重新插入测序芯片，用力向下按压，以确保芯片的连接器引脚与测序仪牢固接触。如问题仍未得到解决，请联系我们的技术支持。 |
未能达到目标温度
| 现象 | 可能原因 | 措施及备注 |
|---|---|---|
| MinKNOW显示“未能达到目标温度” | 测序仪所处环境低于标准室温，或通风不良（以致芯片过热） | MinKNOW会限定测序芯片达到目标温度的时间。当超过限定时间后，系统会显示出错信息，但测序实验仍会继续。值得注意的是，在错误温度下测序可能会导致通量和数据质量（Q值）降低。请调整测序仪的摆放位置，确保其置于室温下、通风良好的环境中后，再在MinKNOW中继续实验。有关MinION温度控制的更多信息，请参考此 FAQ （常见问题）文档。 |
Last updated: 8/23/2024
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