Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
MinION: Protocol
Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114) V ULK_9177_v114_revP_11Dec2024
- Extraction of ultra-high molecular weight (uHMW) gDNA
- Reliably generate and sequence ultra-long read length N50s (>50 kb)
- High yield
- Compatible with R10.4.1 flow cells
For Research Use Only
This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Sample preparation
- 3. Isolation of white blood cells (WBCs) from whole blood
- 4. Preparation of tissue samples for gDNA extraction
- 5. uHMW gDNA extraction
- 6. (Optional) gDNA quantification
Library preparation
- 7. Tagmentation
- 8. Clean-up (using the precipitation star)
- 9. Clean-up (no precipitation star)
- 10. Priming and loading the SpotON flow cell
- 11. Reloading ultra-long DNA library on a MinION/GridION flow cell
测序及数据分析
Troubleshooting
概览
- Extraction of ultra-high molecular weight (uHMW) gDNA
- Reliably generate and sequence ultra-long read length N50s (>50 kb)
- High yield
- Compatible with R10.4.1 flow cells
For Research Use Only
This is an Early Access product For more information about our Early Access programmes, please see this article on product release phases.
1. Overview of the protocol
Introduction to the Ultra-Long DNA Sequencing Kit protocol (SQK-ULK114)
This protocol describes the complete workflow from extracting gDNA from frozen tissue or purified cells from whole blood to the sequencing of ultra-high molecular weight (uHMW) gDNA using the Ultra-Long DNA Sequencing Kit (SQK-ULK114). We have also included the procedure to isolate white blood cells (WBCs) from whole blood and how to quantify gDNA developed by Paul A ‘Giron’ Koetsier & Eric J Cantor, 2021.
We have used the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060) to extract the uHMW gDNA for both input types when developing this protocol. Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.
Per reaction, there is enough library generated for multiple consecutive loads onto a flow cell to increase output. To load a library six times on a MinION/GridION flow cell, a flow cell wash is required to recover channels.
Steps in the sequencing workflow: Prepare for your experiment You will need to:
- If working with whole blood, isolate white blood cells. If working with frozen tissue, isolate cells from the tissue
- Extract your uHMW gDNA
- Quantify your sample
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- If not already installed, download the software for acquiring and analysing your data
- Check your flow cell(s) to ensure it has enough pores for a good sequencing run
Library preparation You will need to:
- Tagment your DNA using a diluted fragmentation mix
- Attach Rapid Adapters to the DNA ends
- Clean-up your sample by precipitating your DNA and elute overnight
- Prime the flow cell and load your DNA library into the flow cell
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
- Optional: Start the EPI2ME software and select a workflow for further analysis
Flow cell loading and flushing
The Ultra-Long DNA Sequencing Kit (SQK-ULK114) protocol generates viscous DNA which can affect flow cell loading. We have modified the flow cell loading steps to take account for this. Please take care and follow the steps carefully to avoid damaging the flow cell.
To increase output, we recommend loading an ultra-long library three times per flow cell. A flow cell wash using the Flow Cell Wash Kit (EXP-WSH004) is required between each subsequent library load to recover channels. To run a second library straight away, please follow the modified method in this protocol: To run another library of ultra-long DNA on a MinION/GridION flow cell straight away.
Best practice for handling uHMW gDNA
When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.
To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.
While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.
For further information, please refer to the troubleshooting section.
重要
Compatibility of this protocol
This protocol should only be used in combination with:
- Ultra-Long DNA Sequencing Kit (SQK-ULK114)
- Flow Cell Wash Kit (EXP-WSH004)
- R10.4.1 flow cells (FLO-MIN114)
- EEB Expansion (EXP-EEB001)
- Ultra-Long Auxiliary Vials (EXP-ULA001)
- MinION Mk1B - MinION Mk1B IT requirements document
- MinION Mk1C - MinION Mk1C IT requirements document
- MinION Mk1D - MinION Mk1D IT requirements document
- GridION - GridION IT requirements document
2. Equipment and consumables
材料
- Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
- Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
- 测序芯片清洗剂盒（EXP-WSH004）
耗材
- MinION 和 GridION测序芯片
- Isopropanol, 100% (Fisher, 10723124)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit™ 分析管（Invitrogen, Q32856）
- （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
- 1.5 ml Eppendorf DNA LoBind离心管
- 2 ml Eppendorf DNA LoBind 离心管
- 5 ml Eppendorf DNA LoBind tubes
- 15 ml Falcon tubes
仪器
- MinION 或 GridION 测序仪
- MinION 及GridION 测序芯片遮光片
- Thermal cycler or heat block
- Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
- 涡旋混匀仪
- 迷你离心机
- Wide-bore pipette tips
- P1000 移液枪和枪头
- P200 移液枪和枪头
- 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
重要
The above list of materials, consumables, and equipment is for the library preparation section of the protocol. Depending on the sample type, additional reagents will be needed for sample processing and DNA extraction; these are listed in the "Sample preparation" section of the protocol.
For this protocol, you will need to extract gDNA from 6 million cells in 40 µl PBS before starting the library preparation.
This protocol has been developed using the NEB Monarch® HMW DNA Extraction Kit for Tissue (cat # T3060). Alternative kits are available from NEB which are specifically designed for the extraction from blood and cells. However, they have not been validated by Oxford Nanopore Technologies.
This method has been validated for use on the following inputs:
- 6 million white blood cells isolated from 1.6 ml blood (bovine), using RBC Lysis Solution (QIAGEN, cat # 158904)
- 6 million cells isolated from 1 g frozen tissue, using pluriSelect Cell Straining equipment.
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。
Ultra-Long DNA Sequencing Kit (SQK-ULK114) contents
We had previously uncovered an issue with the Precipitation Stars (PS) found in the SQK-ULK114 kits and removed their use from the method to prevent any potential issues.
We have since improved our manufacturing and internal validation processes of the Precipitation Stars (PS) and are now in the position to reintroduce their use with the SQK-ULK114 Kits.
Kit format with improved precipitation stars that can be used:
Batch ULK114.30.0001 or newer
| Name | Acronym | Cap colour | Number of vials | Fill volume per vial (µl) |
|---|---|---|---|---|
| Rapid Adapter | RA | Green | 1 | 40 |
| Fragmentation Mix | FRA | Amber | 1 | 50 |
| FRA Dilution Buffer | FDB | Clear | 1 | 1,600 |
| Elution Buffer | EB | Black | 2 | 1,500 |
| Extraction EB | EEB | Orange | 3 | 1,700 |
| Sequencing Buffer UL | SBU | Red | 2 | 1,000 |
| Loading Solution UL | LSU | White cap, pink label | 1 | 200 |
| Flush Tether UL | FTU | Purple | 1 | 600 |
| Flow Cell Flush | FCF | Blue | 2 | 15,500 |
| Precipitation Buffer | PTB | Blue | 2 | 1,700 |
| Precipitation Star | PS | Yellow | 6 | 1 star |
Kit format where stars should not be used:
Batch ULK114.20.xxxx or older:
| Name | Acronym | Cap colour | Number of vials | Fill volume per vial (µl) |
|---|---|---|---|---|
| Rapid Adapter | RA | Green | 1 | 40 |
| Fragmentation Mix | FRA | Amber | 1 | 50 |
| FRA Dilution Buffer | FDB | Clear | 1 | 1,600 |
| Elution Buffer | EB | Black | 2 | 1,500 |
| Extraction EB | EEB | Orange | 3 | 1,700 |
| Sequencing Buffer UL | SBU | Red | 2 | 1,000 |
| Loading Solution UL | LSU | White cap, pink label | 1 | 200 |
| Flush Tether UL | FTU | Purple | 1 | 600 |
| Flow Cell Flush | FCF | Blue | 2 | 15,500 |
| Precipitation Buffer | PTB | Blue | 2 | 1,700 |
| Precipitation Star | PS | Yellow | 6 | 1 star |
Flow Cell Wash Kit (EXP-WSH004) contents
| 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.
To maximise the use of the Ultra-Long DNA Sequencing Kit V14, the EEB Expansion (EXP-EEB001) and the Ultra-Long Auxiliary Vials (EXP-ULA001) expansion packs are available.
These expansions provide extra library preparation and flow cell priming reagents to allow users to maximise the use out of their Ultra-Long DNA Sequencing Kit V14.
The EEB Expansion (EXP-EEB001) contains enough reagents for at least 6 standard extraction elution steps.
The Ultra-Long Auxiliary Vials (EXP-ULA001) provides enough reagents to carry out twelve additional flow cell loads on MinION or PromethION flow cells.
EEB Expansion (EXP-EEB001) contents:
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (μl) |
|---|---|---|---|---|
| Extraction EB | EEB | White | 1 | 6,000 |
Ultra-Long Auxiliary Vials (EXP-ULA001) contents:
| Name | Acronym | Cap colour | No. 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 |
3. Isolation of white blood cells (WBCs) from whole blood
材料
- 1.6 ml of whole blood
耗材
- RBC Lysis Solution (QIAGEN, 158106)
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- 15 ml Falcon tubes
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
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 feel are most appropriate for the whole blood sample to be used. If 6 million cells have been isolated, users can start from the uHMW gDNA extraction step.
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 the cell pellet forward into the "uHMW gDNA extraction" step.
4. Preparation of tissue samples for gDNA extraction
材料
- Cell Suspension Buffer (CSB): 0.35 M sucrose, 100 mM EDTA, 50 mM Tris.HCl pH 8
- Frozen tissue sample
耗材
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- 2.5 M sucrose
- Nuclease-free water (e.g. ThermoFisher, cat #AM9937)
- 50 ml Falcon tubes
- 5 ml Eppendorf DNA LoBind tubes
仪器
- Centrifuge suitable for 5 ml Eppendorf tubes (Eppendorf centrifuge 5804/5804 R or equivalent)
- Eppendorf tube rack suitable for 5 ml Eppendorf tubes
- Scalpel
- TissueRuptor II (QIAGEN, cat # 9002755)
- TissueRuptor Disposable Probes (QIAGEN, cat # 990890)
- Florescent microscope with functionality to quantify nuclei (Logos CELENA S Digital Imaging System or equivalent)
- Heat block equipped with thermoblock suitable for 5 ml Eppendorf tubes
- 200 µm PluriStrainer® (pluriSelect, 43-50200-03)
- 100 µm PluriStrainer® (pluriSelect, 43-50100-51)
- 50 µm PluriStrainer® (pluriSelect, 43-50050-03)
- 30 µm PluriStrainer® (pluriSelect, 43-50030-03)
- PluriStrainer® Connector Ring (pluriSelect, 41-50000-03)
- PluriStrainer® Funnel (pluriSelect, 42-50000)
- P1000 移液枪和枪头
- 10 ml syringe
Prepare the Cell Suspension Buffer (CSB) as follows:
| Reagent | Stock | Final conc. | Volume |
|---|---|---|---|
| Tris.HCl, pH 8 | 1 M | 0.05 M | 50 ml |
| EDTA | 0.5 M | 0.1 M | 200 ml |
| Sucrose | 2.5 M | 0.35 M | 140 ml |
| Nuclease-free water | - | - | 610 ml |
| Total | - | - | 1000 ml |
Add 1 g of the frozen tissue sample to a weighing boat.
Using the scalpel, slice the tissue into thin strips and then dice the sample.
Transfer the tissue sample to a fresh 50 ml Falcon tube.
Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.
Using the QIAGEN TissueRuptor, gently homogenise the tissue sample.
- Insert the probe and pulse at minimum speed for one second. Stir the homogenate between each pulse.
- Repeat this five times.
重要
During homogenisation, only apply as much force as is required to gently break up the tissue. Excessive force will damage the nuclei and make them difficult to quantify. It is not a problem if there is intact material remaining at the end of this step, as it will be re-processed in later steps.
Assemble the pluriStrainer apparatus with a 200 μm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the tissue sample homogenate through the 200 µm PluriStrainer®.
The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.
Disassemble the pluriStrainer® apparatus according to the manufacturer's instructions, setting aside the strained homogenate in the 50 ml Falcon tube for later use.
Repeat the homogenisation process on any intact tissue caught by the pluriStrainer®:
Transfer any intact tissue caught by the 200 µm pluriStrainer® into a fresh 50 ml Falcon tube by inverting the strainer and tapping out the intact tissue.
Tip: A spatula can be used to help remove the intact tissue from the strainer.Add 10 ml of the Cell Suspension Buffer (CSB) into the 50 ml Falcon tube.
Repeat steps 6-10 two more times to perform a total of three rounds of tissue homogenisation.
Combine the contents of the 50 ml Falcon tube with the original strained homogenate set aside in step 10.
CHECKPOINT
The combined volume of 200 µm strained homogenate is ready for further processing.
Strain the 200 µm strained homogenate through the 100 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 100 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 200 µm strained homogenate through the 100 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.
Disassemble the pluriStrainer® and retain the 100 µm strained homogenate in the 50 ml Falcon tube.
Strain the 100 µm strained homogenate through the 50 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 50 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 100 µm strained homogenate through the 50 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.
Disassemble the pluriStrainer® and retain the 50 µm strained homogenate in the 50 ml Falcon tube.
Strain the 50 µm strained homogenate through the 30 µm pluriStrainer®:
Assemble the pluriStrainer apparatus with a 30 µm strainer, connector ring, funnel and 50 ml Falcon tube according to the manufacturer’s instructions.
Pass the full volume of the 50 µm strained homogenate through the 30 µm PluriStrainer®. Tip: The homogenate can be gently agitated, and a small amount of negative pressure can be applied with the syringe to help pass the homogenate through the strainer.
Disassemble the pluriStrainer® and retain the 30 µm strained homogenate in the 50 ml Falcon tube.
Determine the concentration of the nuclei in the purified homogenate using a fluorescent microscope and a stain appropriate for the nuclei in the sample.
Take forward a volume corresponding to 6 million nuclei and add this to a 5 ml Eppendorf DNA LoBind tube.
Centrifuge the 5 ml Eppendorf tube at 16,000 x g for five minutes to pellet the nuclei/cells.
Pipette off all the supernatant and discard, taking care not to disturb the pellet.
Add 40 µl of PBS to the 5 ml Eppendorf DNA LoBind tube.
Thoroughly mix the tube by repeatedly flicking. Ensure the pellet breaks up and no clumps remain in the nuclei/cell suspension.
Note: You may need to flick quite hard and thoroughly to ensure the pellet breaks up and no clumps remain.
步骤结束
Take the nuclei/cell suspension forward into the "uHMW gDNA extraction" step.
5. uHMW gDNA extraction
材料
- 6 million cells/nuclei isolated from frozen tissue or white blood cells isolated from whole blood
- Extraction EB (EEB)
- Monarch® HMW DNA Extraction Kit for Tissue (New England Biolabs, T3060)
耗材
- 5 ml Eppendorf DNA LoBind tubes
- Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
- Isopropanol, 100% (Fisher, 10723124)
- Ethanol, 100% (e.g. Fisher, 16606002)
- 2 ml Eppendorf DNA LoBind 离心管
仪器
- Heat block set at 56°C
- Thermomixer set at 56°C (suitable for 1.5 ml, 2 ml and 5 ml tubes)
- Hula混匀仪（低速旋转式混匀仪）
- 迷你离心机
- P1000 pipette and 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
- Eppendorf 5424 离心机（或等效器材）
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.
Add 6 million cells resuspended in 40 µl PBS to a fresh 5 ml tube. Cells can be isolated from cell culture, white blood cells from blood, or tissue according to the above methods.
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.
提示
When using cell lines, we have found that the incubation step below can be omitted.
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.
提示
When using cell lines, we have found the protein removal steps can be omitted. If using cell lines, proceed directly to step 13.
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 (or to the lysis reaction if proceeded directly from Step 9).
Note: The first bead is a sacrificial bead and will remain 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.
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.
Transfer the beads to a Monarch Bead Retainer inserted in a Monarch Collection Tube II.
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.
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.
步骤结束
Take forward the resuspended DNA into the quantification step. However, at this point it is possible to store the sample at room temperature overnight.
6. (Optional) gDNA quantification
材料
- Monarch® DNA Capture Beads
- Monarch® Bead Retainer
- Monarch® Collection Tubes II
耗材
- 2 ml Eppendorf DNA LoBind 离心管
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
仪器
- 涡旋混匀仪
- Centrifuge
- Qubit荧光计 （或用于质控检测的等效仪器）
- P200 移液枪和枪头
Quantification of uHMW gDNA
The method to quantify uHMW gDNA was developed by Paul A ‘Giron’ Koetsier & Eric J Cantor, 2021, which recommends the use of a regular P200 pipette and tip.
This optional uHMW gDNA quantification step has also been included in the protocol for user QC. However, this step can be omitted and 750 µl of DNA in Extraction EB (EEB) can be taken straight into the tagmentation step of the protocol.
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.
Using a wide-bore pipette tip, transfer the gDNA into a clean Eppendorf DNA LoBind tube.
The size of Eppendorf DNA LoBind tube depends on if you are using the Precipitation Star (PS) during the clean-up step:
Clean-up using the Precipitation Star (PS)
- Transfer the gDNA into a clean 1.5 ml Eppendorf DNA LoBind tube.
Clean-up not using the Precipitation Star (PS):
- Transfer the gDNA into a clean 2 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 forward 750 µl DNA into the tagmentation step.
7. Tagmentation
材料
- 750 µl of extracted uHMW gDNA in EEB
- 快速测序文库接头（RA）
- Fragmentation Mix (FRA)
- FRA Dilution Buffer (FDB)
耗材
- 1.5 ml Eppendorf DNA LoBind离心管
仪器
- Thermal cycler or heat block
- 迷你离心机
- P1000 pipette and wide-bore pipette tips
- P1000 移液枪和枪头
- P20 移液枪和枪头
- Ice bucket with ice
Best practice for handling uHMW gDNA
When mixing, we recommend using wide-bore pipette tips to mix the full volume of a sample to ensure thorough mixing whilst minimising mechanical shearing of long fragments.
To preserve longer DNA, mix slower and more gently. Vortexing on low speeds may also be used at the expense of very long fragments.
While precautions should be taken to ensure that DNA fragment lengths are preserved, there should be no compromise to ensuring that reagents are thoroughly mixed with DNA. Insufficient mixing will lead to reduced read length and output.
For further information, please refer to the troubleshooting section.
Thaw the the kit components at room temperature, spin down briefly using a microfuge and mix by pipetting as indicated by the table below:
Once thawed, keep all the kit components on ice.
| Reagent | Thaw at room temperature | Briefly spin down | Mix well by pipetting |
|---|---|---|---|
| Fragmentation Mix (FRA) | Not frozen | ✓ | ✓ |
| FRA dilution buffer (FDB) | Not frozen | ✓ | ✓ |
| Rapid Adapter (RA) | Not frozen | ✓ | ✓ |
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.
8. Clean-up (using the precipitation star)
材料
- Elution Buffer from the Oxford Nanopore kit (EB)
- Precipitation Buffer (PTB)
- Precipitation Star (PS)
耗材
- 1.5 ml Eppendorf DNA LoBind 离心管
仪器
- Centrifuge
- Microfuge
- Hula混匀仪（低速旋转式混匀仪）
- P200 移液枪和枪头
- P1000 移液枪和枪头
- P1000 pipette and wide-bore pipette tips
重要
Ensure you are using the correct method depending on your batch of Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114).
We had previously communicated an issue with the Precipitation Star (PS) found in the SQK-ULK114 kits and updated the protocol to address it.
We have since improved our manufacturing and internal validation processes and are now in the position to reintroduce the use of the Precipitation Star (PS) within the SQK-ULK114 protocol.
| Kit format where stars should not be used: | Kit format with improved precipitation stars that can be used: |
|---|---|
Batch ULK114.20.xxxx or older  | Batch ULK114.30.0001 or newer  |
Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by vortexing as indicated by the table below:
| Reagent | Thaw at room temperature | Briefly spin down | Mix well by pipetting |
|---|---|---|---|
| Precipitation buffer (PTB) | ✓ | ✓ | ✓ |
| Elution Buffer (EB) | ✓ | ✓ | ✓ |
Once thawed, keep all the kit components on ice.
提示
We strongly recommend using a 1.5 ml Eppendorf DNA LoBind tube for the clean-up method when using the Precipitation Star (PS)
Add a Precipitation Star (PS) to the sample.
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 around the Precipitation Star (PS).
Using a regular pipette tip, carefully remove the supernatant from the tube, taking care not to aspirate the DNA.
In the 1.5 ml Eppendorf LoBind DNA tube, the Precipitation Star (PS) should be suspended mid-way in the tube, allowing the supernatant beneath the star to be removed.
Briefly spin down the tube and remove any residual supernatant using a regular pipette tip, taking care not to aspirate the DNA.
Using a regular pipette tip, add 300 µl of Elution Buffer (EB) to the tube containing the Precipitation Star (PS) and DNA. Incubate overnight at room temperature, for a minimum of 12 hours.
Using a wide-bore pipette tip, remove and retain eluate containing the DNA library into a clean 1.5 ml Eppendorf LoBind DNA tube.
Briefly spin down the tube containing the Precipitation Star (PS) and remove any remaining eluate with a wide-bore pipette tip, ensuring there is no liquid remaining on the Precipitation Star (PS).
Dispose of the tube containing the Precipitation Star (PS).
Gently mix the DNA library by slowly pipetting 10 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
Take the DNA library forwards for loading into the flow cell. Store the library on ice until ready to load.
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。
9. Clean-up (no precipitation star)
材料
- Elution Buffer from the Oxford Nanopore kit (EB)
- Precipitation Buffer (PTB)
耗材
- 2.0 ml Eppendorf DNA LoBind tubes
仪器
- Centrifuge
- Microfuge
- Hula混匀仪（低速旋转式混匀仪）
- P200 移液枪和枪头
- P1000 移液枪和枪头
- P1000 pipette and wide-bore pipette tips
重要
Ensure you are using the correct method depending on your batch of Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114).
We had previously communicated an issue with the Precipitation Star (PS) found in the SQK-ULK114 kits and updated the protocol to address it.
We have since improved our manufacturing and internal validation processes and are now in the position to reintroduce the use of the Precipitation Star (PS) within the SQK-ULK114 protocol.
| Kit format where stars should not be used: | Kit format with improved precipitation stars that can be used: |
|---|---|
| Batch ULK114.20.xxxx or older | Batch ULK114.30.0001 or newer |
Thaw the kit components at room temperature, spin down briefly using a microfuge and mix by vortexing as indicated by the table below:
| Reagent | Thaw at room temperature | Briefly spin down | Mix well by pipetting |
|---|---|---|---|
| Precipitation buffer (PTB) | ✓ | ✓ | ✓ |
| Elution Buffer (EB) | ✓ | ✓ | ✓ |
Once thawed, keep all the kit components on ice.
提示
We recommend using a 2 ml Eppendorf DNA LoBind tube for the clean-up method when not using the Precipitation Star (PS).
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 300 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 10 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
Take the DNA library forwards for loading into the flow cell. Store the library on ice until ready to load.
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。
10. Priming and loading the SpotON flow cell
材料
- 测序芯片冲洗液（FCF）
- Flush Tether UL (FTU)
- Loading Solution UL (LSU)
- Sequencing Buffer UL (SBU)
耗材
- 1.5 ml Eppendorf DNA LoBind 离心管
- MinION 和 GridION测序芯片
- （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
仪器
- MinION 或 GridION 测序仪
- MinION 及GridION 测序芯片遮光片
- P1000 pipette and wide-bore pipette tips
- P200 pipette and 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
重要
Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).
Only use the reagents provided with the SQK-ULK114 kit for priming and loading the flow cell. Reagents from other kits are not compatible with this protocol.
重要
Ensure the MinION Flow Cell Light Shield is not installed on to your flow cell until after loading your DNA library.
To ensure optimal interactions and access to the flow cell ports, please ensure the MinION Flow Cell Light Shield is not installed on your flow cell until after loading your DNA library.
If the MinION Flow Cell Light Shield has already been installed, remove it from the flow cell and store until required later in the protocol.
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 new tube, prepare the DNA library for loading as follows. Use a wide-bore pipette tip for the addition of the DNA library:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer UL (SBU) | 37.5 µl |
| Loading Solution UL (LSU) | 3.7 µl |
| DNA library | 33.8 µl |
| Total | 75 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.
打开MinION或GridION测序仪的盖子，将测序芯片插入金属固定夹的下方。用力向下按压芯片，以确保正确的热、电接触。
顺时针转动预处理孔孔盖，使预处理孔显露出来。
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：
- 将P1000移液枪转至200µl刻度。
- 将枪头垂直插入预处理孔中。
- 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。
__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。
重要
For optimal sequencing performance and improved output on R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml. We do not recommend using recombinant BSA.
For optimal sequencing performance and improved output on R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml. We do not recommend using recombinant BSA.
To prepare the flow cell priming mix with BSA, combine the following reagents in a 1.5 ml Eppendorf tube, and mix by inverting the tube and pipette mix at room temperature:
| Reagent | Volume |
|---|---|
| Bovine Serum Albumin (BSA) at 50 mg/ml | 5 µl |
| Flush Tether UL (FTU) | 30 µl |
| Flow Cell Flush (FCF) | 1170 µl |
| Total | 1205 µl |
重要
Ensure the MinION Flow Cell Light Shield is not installed on the flow cell at this stage.
If the MinION Flow Cell Light Shield has already been installed, remove it from the flow cell and store until required later in the protocol.
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes.
完成测序芯片的预处理：
- 轻轻地翻起SpotON上样孔盖，使SpotON上样孔显露出来。
- 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。
Ensure the SpotON port and Priming port covers of the flow cell are open in preparation for loading.
Using a wide-bore pipette tip, load the DNA library onto the SpotON port until 75 μl has been loaded.
Take care not to place the pipette tip directly onto/into the SpotON port as this could damage the array.
Allow the DNA library to flow through the SpotON port by waiting up to two minutes.
If the DNA library does not enter the SpotON port, apply negative pressure in the flow cell as explained further below.
Cover Waste port 2 and the Priming port with clean, gloved fingers.
Using a fully depressed P200 pipette, insert the tip in Waste port 1 whilst Waste port 2 and the Priming port are covered.
Very slowly aspirate to pull the DNA library into the SpotON sample port. Closely watch the DNA library on the SportON port and completely remove the pipette as soon as the library starts to be pulled into the port.
Note: Take care to not apply too much negative pressure too quickly to avoid bringing air bubbles into the flow cell. Air bubbles will cause irreversible damage to the flow cell.
轻轻合上SpotON加样孔孔盖，确保塞头塞入加样孔内。逆时针转动预处理孔孔盖，盖上预处理孔。
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。
按下述步骤安装测序芯片遮光片：
小心将遮光片的前沿（平端）与金属固定夹的边沿对齐。 请注意： 请勿将遮光片强行压到固定夹下方。
将遮光片轻轻盖在测序芯片上。遮光片的SpotON加样孔孔盖缺口应与芯片上的SpotON加样孔孔盖接合，遮盖住整个测序芯片的前部。
注意
MinION测序芯片的遮光片并非固定在测序芯片上，因此当为芯片加装遮光片后，请小心操作。
步骤结束
小心合上测序设备上盖并在MinKNOW上设置测序实验。
We recommend loading an ultra-long DNA library three times per flow cell to increase output.
A nuclease wash using the Flow Cell Wash Kit (EXP-WSH004) is required between each subsequent library load to recover channels and maximise sequencing output.
For MinION/GridION flow cells, there is enough library generated for six consecutive loads per reaction, using 33.8 µl of fresh library combined with 37.5 µl of Sequencing Buffer (SBU) and 3.7 µl of Loading Solution (LSU) before re-loading for further sequencing.
Please follow Flushing a MinION/GridION Flow Cell in the Flow Cell Wash Kit protocol for the nuclease wash instructions. To run another library straight away, follow the instructions below.
11. Reloading ultra-long DNA library on a MinION/GridION flow cell
材料
- 测序芯片清洗剂盒（EXP-WSH004）
- Flush Tether UL (FTU)
- 测序芯片冲洗液（FCF）
- Sequencing Buffer UL (SBU)
- Loading Solution UL (LSU)
耗材
- 1.5 ml Eppendorf DNA LoBind离心管
- （非必需）牛血清白蛋白（BSA）（50 mg/mL）（例如 Invitrogen™ UltraPure™ BSA （50 mg/mL）， AM2616）
仪器
- P1000 pipette and wide-bore pipette tips
- P200 pipette and 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
重要
Before reloading your library or loading a new library, please ensure you wash the flow cell using the Flow Cell Wash Kit (EXP-WSH004).
Follow the instructions in the Flow Cell Wash Kit (EXP-WSH004) for MinION/GridION protocol.
- This washing procedure aims to remove most of the initial library and prepare the flow cell for loading of a subsequent library.
- Data acquisition in MinKNOW should be paused during the wash procedure and library loading.
- After the flow cell has been washed, another library can be loaded.
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.
To run a second library of ultra-long DNA straight after flushing a flow cell, we recommend removing all fluid from the waste channel after each priming step.
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 new tube, prepare the DNA library for loading as follows. Use a wide-bore pipette tip for the addition of the DNA library:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer UL (SBU) | 37.5 µl |
| Loading Solution UL (LSU) | 3.7 µl |
| DNA library | 33.8 µl |
| Total | 75 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.
Slide the priming port cover of the flow cell clockwise to open the priming port.
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：
- 将P1000移液枪转至200µl刻度。
- 将枪头垂直插入预处理孔中。
- 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。
__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。
重要
为在MinION及GridION R10.4.1测序芯片（FLO-MIN114）上获得最优的测序表现并提高测序产出，我们推荐您向测序芯片预处理液中加入终浓度为0.2 mg/ml的牛血清白蛋白（BSA）。
请注意： 我们不推荐使用其它类型的白蛋白（例如重组人血清白蛋白）。
To prepare the flow cell priming mix with BSA, combine the following reagents in a 1.5 ml Eppendorf tube, and mix by inverting the tube and pipette mix at room temperature:
| Reagent | Volume |
|---|---|
| Bovine Serum Albumin (BSA) at 50 mg/ml | 5 µl |
| Flush Tether UL (FTU) | 30 µl |
| Flow Cell Flush (FCF) | 1170 µl |
| Total | 1205 µl |
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes.
重要
It is vital to wait five minutes between the priming mix flushes to ensure effective removal of the nuclease.
Close the priming port cover and ensure the SpotON sample port cover is closed.
重要
It is vital that the flow cell priming port and SpotON sample port are closed before removing the waste buffer to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.
Remove the waste buffer, as follows:
- Close the priming port and SpotON sample port cover, as indicated in the figure below.
- Insert a P1000 pipette into waste port 1 and remove the waste buffer.
Note: As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
Slide open the priming port and load 200 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles.
Close the priming port and use a P1000 to remove all fluid from the waste channel through Waste Port 1.
Ensure the SpotON port and Priming port covers of the flow cell are open in preparation for loading.
Using a wide-bore pipette tip, load the DNA library onto the SpotON port until 75 μl has been loaded.
Take care not to place the pipette tip directly onto/into the SpotON port as this could damage the array.
Allow the DNA library to flow through the SpotON port by waiting up to two minutes.
If the DNA library does not enter the SpotON port, apply negative pressure in the flow cell as explained further below.
Cover Waste port 2 and the Priming port with clean, gloved fingers.
Using a fully depressed P200 pipette, insert the tip in Waste port 1 whilst Waste port 2 and the Priming port are covered.
Very slowly aspirate to pull the DNA library into the SpotON sample port. Closely watch the DNA library on the SportON port and completely remove the pipette as soon as the library starts to be pulled into the port.
Note: Take care to not apply too much negative pressure too quickly to avoid bringing air bubbles into the flow cell. Air bubbles will cause irreversible damage to the flow cell.
轻轻合上SpotON加样孔孔盖，确保塞头塞入加样孔内。逆时针转动预处理孔孔盖，盖上预处理孔。
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。
按下述步骤安装测序芯片遮光片：
小心将遮光片的前沿（平端）与金属固定夹的边沿对齐。 请注意： 请勿将遮光片强行压到固定夹下方。
将遮光片轻轻盖在测序芯片上。遮光片的SpotON加样孔孔盖缺口应与芯片上的SpotON加样孔孔盖接合，遮盖住整个测序芯片的前部。
注意
MinION测序芯片的遮光片并非固定在测序芯片上，因此当为芯片加装遮光片后，请小心操作。
Once the flow cell is reloaded, resume the sequencing run on MinKNOW and trigger a pore scan.
To resume sequencing run, navigate to the Experiments page, click 'Resume' and select flow cell position.
To manually trigger a channel scan, click 'Start pore scan' and select flow cell position.
For further information, please see the MinKNOW protocol.
Resume run:
Pore scan:
12. 数据采集和碱基识别
如何开始测序
在完成测序芯片的加样后，您即可在MinKNOW中启动测序实验。MinKNOW 软件负责仪器控制、数据采集以及实时碱基识别。有关设置和使用 MinKNOW 的详细信息，请参阅MinKNOW 实验指南。
您可以通过多种方式使用并设置MinKNOW：
- 在直接或远程连接到测序设备的计算机上。
- 直接在 GridION、MinION Mk1C 或 PromethION 24/48 测序设备上。
有关在测序设备上使用 MinKNOW 的更多信息，请参阅相应设备的用户手册：
在MinKNOW中启动测序：
1. 在 "开始 "（Start）页面上，选择 __开始测序__（Start Sequencing)。 
2. 输入实验详情：例如实验名称，测序芯片位置及样本ID。 
3. 在"试剂盒"页面上，选择建库试剂盒。 
4. 配置测序实验参数，或保持“运行选项”和“分析”页面中的默认设置。
请注意： 如果在设置运行参数时关闭了碱基识别，您可在实验结束后，在MinKNOW中运行线下碱基识别。详情请参阅MinKNOW实验指南。
5. 在“输出”页面中，设置输出参数或保持默认设置。 
6. 单击 "参数确认" 页面上的 开始 启动测序。 
测序后数据分析
当于MinKNOW上完成测序后，您可按照“测序芯片的重复利用及回收”一节中的说明重复使用或返还测序芯片。
完成测序和碱基识别后，即可进行数据分析。有关碱基识别和后续分析选项的详细信息，请参阅数据分析文档。
在下游分析部分，我们将概述更多用于数据分析的选项。
13. 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。
14. 下游分析
下游分析
您可以选择以下几个途径来进一步分析经过碱基识别的数据：
1. EPI2ME 工作流程
Oxford Nanopore Technologies通过EPI2ME提供了一系列针对高阶数据分析的生物信息学教程和工作流程。上述资源汇总于纳米孔社区的 EPI2ME 板块。该平台通过描述性文字、生物信息学代码和示例数据，具象化地展示出我们的研究和应用团队发布在 GitHub 上的工作流程。
2. 科研分析工具
Oxford Nanopore Technologies的研发部门开发了许多分析工具，您可在Oxford Nanopore的 GitHub 资料库中找到。这些工具面向有一定经验的用户，并包含如何安装和运行软件的说明。工具以源代码形式提供，因此我们仅提供有限的技术支持。
3. 纳米孔社区用户开发的分析工具
如上述资源未能提供满足您研究需求的数据分析方法，请前往资源中心，查找适用的生物信息学工具。该板块汇总了许多由纳米孔社区成员开发、且在Github上开源的、针对纳米孔数据的生信分析工具。请注意，Oxford Nanopore Technologies不为这些工具提供支持，也不能保证它们与测序所用的最新的化学试剂/软件配置兼容。
15. Issues during 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。
Troubleshooting
| Observation | Comments and actions |
|---|---|
| Low throughput | 1. Vortex gently after adding the diluted Fragmentation Mix (FRA) to break up the largest fragments. 2. Ensure the diluted Fragmentation Mix (FRA) is thoroughly mixed with the gDNA. 3. Use less input material if the DNA library was too viscous to load onto the flow cell. |
| DNA is too viscous and will not load onto a flow cell | 1. Lower the input material to reduce the amount of gDNA going into the library preparation and reduce viscosity. 2. If DNA library will not load using the method outlined in this protocol, slowly pipette mix 5 times with a standard P200 pipette set to the full volume of the library and reload the flow cell. |
| Read lengths are not long enough | 1. Increase input material. Note: Library viscosity increases with more gDNA. 2. Reduce volume of Fragmentation Mix (FRA) added to FRA Dilution Buffer (FDB) to avoid over-fragmentation of gDNA. Note: We do not recommend diluting less than 2 µl Fragmentation Mix (FRA). 3. We recommend using PFGE to check the extracted gDNA is of ultra-high molecular weight (uHMW), thus capable of generating ultra-long read lengths. |
| No sequencing output | 1. Check gDNA has been recovered in library preparation using a NanoDrop spectrophotometer. 2. Check viscosity of the sample. The library should be viscous if it contains uHMW gDNA in this protocol. |
| Aspirating supernatant when the DNA has precipitated | Take care to not aspirate the DNA. Remove smaller volumes of supernatant incrementally to reduce the risk of aspirating the DNA. |
| Mixing | Mix slowly and carefully to prevent DNA shearing. Low vortexing can be used to mix at the expense of ultra-long reads. With vortexing, long read lengths of ~90 kb N50 can still be generated with improved outputs. |
| No DNA recovered from the library preparation clean-up | If the DNA is no longer viscous or the NanoDrop reading is low, DNA may have been lost during the clean-up step of the library preparation. 1. Ensure uHMW DNA is used or users risk DNA loss. 2. Take care to not aspirate the precipitated DNA during the clean-up step. To avoid this, remove smaller volumes of supernatant incrementally. Ensure as much supernatant is removed as possible. |
Last updated: 12/11/2024
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