Ligation sequencing V14 â Human cfDNA singleplex (SQK-LSK114)
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PromethION: Protocol
Ligation sequencing V14 â Human cfDNA singleplex (SQK-LSK114) V CFS_9207_v114_revC_15May2024
Method outlining sample extraction, library preparation, sequencing and data analysis. This protocol:
- uses human cfDNA
- is for singleplex sequencing
- is compatible with R10.4.1 flow cells
For Research Use Only
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
Sample preparation
Library preparation
- 4. DNA repair and end-prep
- 5. Adapter ligation and clean-up
- 6. Priming and loading the PromethION Flow Cell
Sequencing and data analysis
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æŠèŠ
Method outlining sample extraction, library preparation, sequencing and data analysis. This protocol:
- uses human cfDNA
- is for singleplex sequencing
- is compatible with R10.4.1 flow cells
For Research Use Only
1. Overview of the protocol
Introduction to the singleplex human cfDNA sequencing protocol
This protocol describes how to carry preparation and sequencing of a human cfDNA sample using the Ligation Sequencing Kit V14 (SQK-LSK114). Typically, we obtain ~50 Gb of aligned data (15x coverage) for human cfDNA samples processed with this protocol.
Prior to library preparation, the sample extraction is carried out using the QIAGEN QIAamp MinElute ccfDNA Midi Kit and following our Human blood cell-free DNA (cfDNA) extraction for singleplex sequencing method.
Note: We recommend that blood samples are processed while fresh, as we have observed potential gDNA contamination arising from blood that has been stored in certain types of collection tubes.
For more information on the development and performance of this method, please refer to our Updated method for cell-free DNA (cfDNA) methylation profiling know-how document. An additional know-how document is also available for the optimisation of library preparation for longer cell-free DNA (cfDNA).
Steps in the workflow
Prepare for your experiment
You will need to:
- Extract your DNA, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- 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 cfDNA from your human blood sample, and quantify the DNA:
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 the cfDNA and prepare the DNA ends for adapter attachment | 125 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 basecall reads.
- (Optional) Raw sequencing data can be basecalled and aligned to a reference using dorado.
- (Optional) Start the EPI2ME software and select a bioinformatics workflow to analyse your data. Alternatively, external tools can be used to further analyse and explore your data.
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Compatibility of this protocol
This protocol should only be used in combination with:
- Ligation Sequencing Kit V14 (SQK-LSK114)
- R10.4.1 PromethION Flow Cells (FLO-PRO114M)
- Flow Cell Wash Kit (EXP-WSH004)
- PromethION 24/48 device - PromethION IT requirements document
- PromethION 2 Solo device - PromethION 2 Solo IT requirements document
2. Equipment and consumables
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- (FOR EXTRACTION) 10 ml of human blood in EDTA K2 vacuum tube, or 3â5 ml of plasma
- (FOR LIBRARY PREPARATION) 15â30 ng extracted human cfDNA per sample
- Ligation Sequencing Kit V14 (SQK-LSK114)
æ¶èå
- PromethION Flow Cell
- QIAamp MinElute ccfDNA Midi Kit (QIAGEN, 55284)
- Agencourt AMPure XP beads (Beckman Coulterâ¢, A63881)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
- NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Isopropanol
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Qubit⢠Assay Tubes (Invitrogen, Q32856)
- 5 ml Eppendorf DNA LoBind tubes
- 15 ml Falcon tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml èå£ã®PCRãã¥ãŒã
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- PromethION device
- PromethION Flow Cell Light Shield
- Centrifuge with capacity for 5 ml and 15 ml tubes, and a swing out and fixed angle rotors
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- Magnetic rack for 15 ml tubes
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- Thermomixer, or other shaker for microcentrifuge tubes, with capacity to heat at 56°C
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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 this protocol, the following inputs are required:
Input requirements per sample for the extraction method:
- 10 ml of fresh human blood in EDTA K2 vacuum tube, or 3â5 ml of plasma
Note: We recommend that blood samples are processed while fresh, as we have observed potential gDNA contamination arising from blood that has been stored in certain types of collection tubes.
Input requirements per sample for the library preparation:
- 15â30 ng extracted human cfDNA
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Flongle Flow Cell | 50 |
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Ligation Sequencing Kit V14 (SQK-LSK114) ã®ã³ã³ãã³ã
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3. Sample extraction method for singleplex sequencing of human cfDNA
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- 10 ml of fresh human blood in EDTA K2 vacuum tube, or 3-5 ml of plasma
æ¶èå
- QIAamp MinElute ccfDNA Midi Kit (QIAGEN, 55284)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Isopropanol
- nuclease-free waterã§èª¿æŽãã 80% ãšã¿ããŒã«æº¶æ¶²
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Qubit⢠Assay Tubes (Invitrogen, Q32856)
- 5 ml Eppendorf DNA LoBind tubes
- 15 ml Falcon tubes
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
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- Centrifuge with capacity for 5 ml and 15 ml tubes, and a swing out and fixed angle rotors
- Microfuge
- Hula mixerïŒç·©ããã«å転ãããããµãŒïŒ
- Magnetic rack for 15 ml tubes
- Magnetic rack
- ãã«ããã¯ã¹ãããµãŒ
- P1000 pipette and tips
- P100 ãããããšããã
- Thermal cycler
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
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- Agilent Femto Pulse System (or equivalent for read length QC)
Optimised extraction: Human blood cell-free DNA (cfDNA) extraction for singleplex sequencing
This extraction method can also be found in the Extraction Protocols tab in the Documentation space on the Nanopore Community: Human blood cell-free DNA (cfDNA) extraction for singleplex sequencing.
These instructions describe a method to extract cell-free DNA (cfDNA) from human blood samples collected in EDTA K2 vacuum tubes (step 1), or human plasma (step 3). Once samples have undergone the necessary centrifugation steps, the extraction is performed using the QIAGEN QIAamp MinElute ccfDNA Midi Kit.
Note: The yield, DIN and sequencing read length of extracted DNA may vary depending on initial sample quality. Please ensure you are following the correct method and using high-quality sample inputs.
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Alternatively, if you have previously extracted and stored your cfDNA sample(s), this can be used directly in the Library preparation section of this protocol.
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We recommend that blood samples are processed while fresh, as we have observed potential gDNA contamination arising from blood that has been stored in certain types of collection tubes.
Preparation of plasma from fresh blood
Centrifuge 10 ml of fresh blood (overnight chilled delivery) in the EDTA K2 vacuum tube at 1,900 x g for 10 minutes at 4°C in a swing out rotor centrifuge.
Pipette and transfer the supernatant (this is the plasma fraction) to a fresh 5 ml DNA LoBind Eppendorf tube.
The volume should be around 3.5â4 ml.
To remove residual cells from the plasma, centrifuge the plasma at 16,000 x g for 10 minutes (or 6,000 x g for 30 minutes depending on the spin capacity of the centrifuge) at 4°C, in a fixed angle rotor.
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It is important to remove residual cells from the sample when the blood/plasma is still fresh (from an overnight chilled delivery). Failing to do so will result in increased amounts of gDNA contamination in the sequencing library.
Aspirate the supernatant and transfer it to a fresh 15 ml tube.
Purification of cfDNA from 3â5 ml serum or plasma Using the QIAamp MinElute ccfDNA Midi Kit
Before starting the extraction:
- Prepare a shaker for microcentrifuge tubes at room temperature for use in step 14.
- Preheat a second shaker at 56°C for use in step 26. (Alternatively, equilibrate the first shaker to 56°C after step 14).
- Resuspend Magnetic Bead Suspension (from the QIAGEN QIAamp MinElute ccfDNA Midi Kit) by pulse-vortexing for 1 min.
Note: Do not let the suspension settle for more than 2 min before use. Pipette from the centre of the suspension.
Prepare the buffers for extraction:
- Add 8 ml isopropanol (100%) to 12 ml Buffer ACB concentrate to obtain 20 ml Buffer ACB. Mix well after adding isopropanol.
- Add 30 ml ethanol (96â100%) to 13 ml Buffer ACW2 concentrate to obtain 43 ml Buffer ACW2. Mix well after adding ethanol.
Mix the following components according to the instructions below in a 15 ml tube:
Component | Volume for 3 ml plasma (µl) | Volume for 4 ml plasma (µl) | Volume for 5 ml plasma (µl) |
---|---|---|---|
Plasma | 3,000 | 4,000 | 5,000 |
Magnetic Bead Suspension | 90 | 120 | 150 |
Proteinase K | 165 | 220 | 275 |
Bead Binding Buffer | 450 | 600 | 750 |
Total volume | 3,705 | 4,940 | 6,175 |
Incubate the reaction for 10 min at room temperature while shaking (at a slow speed) end-over-end.
Spin the tube down briefly (30 seconds at 200 x g) to remove any solution in the cap.
Place the tube containing bead solution into a magnetic rack for 15 ml tubes. Let the tube stand for at least 1 min, until the solution is clear.
Remove and discard supernatant.
Remove the tube from the magnetic rack and add 200 µl of Bead Elution Buffer to the bead pellet. Vortex to resuspend beads, and pipette up and down to mix and rinse residual beads from the tube wall.
Transfer the full volume of mixture (including the beads) into a Bead Elution Tube.
Incubate for 5 min on a shaker for microcentrifuge tubes at room temperature and 300 rpm.
Note: If the same shaker for microcentrifuge tubes is to be used in step 26, remove the tubes after the room temperature incubation and equilibrate the shaker to 56°C
Place the Bead Elution Tube containing the bead solution into a magnetic rack for 2 ml tubes. Let the tube stand for at least 1 min, until the solution is clear.
Transfer the supernatant into a new Bead Elution tube. Discard the bead pellet.
Avoid transferring any magnetic beads in this step. Carryover may result in reduced cfDNA yield.
Add 300 µl Buffer ACB to the Bead Elution tube containing the supernatant, and vortex to mix. Briefly centrifuge the tube to remove drops from inside the lid.
Pipet the supernatantâBuffer ACB mixture from the previous step into a QIAamp UCP MinElute column.
Centrifuge for 1 min at 6,000 x g.
Place the QIAamp UCP MinElute column into a clean 2 ml collection tube, and discard the flow-through.
Add 500 µl Buffer ACW2 to the QIAamp UCP MinElute column.
Centrifuge for 1 min at 6,000 x g.
Place the QIAamp UCP MinElute column into a clean 2 ml collection tube, and discard the flow-through.
Centrifuge the QIAamp UCP MinElute column at 20,000 x g for 3 min.
Place the QIAamp UCP MinElute column into a new 1.5 ml elution tube and discard the 2 ml collection tube.
Open the lid of the tube and incubate the assembly in a shaker for microcentrifuge tubes at 56°C for 3 min to dry the membrane completely.
Carefully pipet 50 µl of ultra-clean water into the centre of the membrane. Close the lid and incubate at room temperature for 1 min.
Centrifuge at 20,000 x g for 1 min to elute the DNA.
To maximise yield from the elution: Place the QIAamp UCP MinElute column in a clean 1.5 ml elution tube. Aspirate the eluate from the previous step and reload it onto the centre of the membrane. Close the lid and incubate 1 min at room temperature.
Centrifuge at 20,000 x g for 1 min to elute the DNA.
Quantify 1 µl of eluted sample using a Qubit fluorometer.
From 3.5â4 ml of plasma, you can expect a yield of between 15â30 ng cfDNA.
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We recommend that the fragment length profiles of extracted cfDNA samples are analysed using a Femto Pulse (Agilent), or equivalent:
Fragment length profile of extracted cfDNA, run on a Femto Pule (Agilent). This example shows the characteristic nucleosome peaks with minimal gDNA contamination.
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Take forward 15â30 ng of extracted human cfDNA to the library preparation stage of the protcol.
4. DNA repair and end-prep
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- 15â30 ng extracted human cfDNA per sample
- AMPure XP Beads (AXP)
æ¶èå
- NEBNext FFPE DNA Repair v2 Module (NEB, E7360)
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
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- Qubit⢠Assay Tubes (Invitrogen, Q32856)
- 0.2 ml èå£ã®PCRãã¥ãŒã
- 1.5 ml Eppendorf DNA LoBind tubes
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- P1000 ããããåã³ããã
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Prepare the NEBNext FFPE DNA Repair Mix, the NEBNext FFPE DNA Repair Buffer v2 and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturerâs instructions, and place on ice.
For optimal performance, NEB recommend the following:
Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed.
Note: Do not vortex the FFPE DNA Repair Mix, NEB Thermoliable Proteinase K or Ultra II End Prep Enzyme Mix.Always spin down tubes before opening for the first time each day.
The FFPE DNA Repair Buffer v2 may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.The FFPE DNA Repair Buffer v2 may have a yellow tinge and is fine to use if yellow.
Prepare the DNA in nuclease-free water:
Ensure you have 15â30 ng of extracted cfDNA from the sample extraction, and transfer this into a 0.2 ml thin-walled PCR tube.
Adjust the volume to 46 ÎŒl with nuclease-free water.
Mix thoroughly by pipetting up and down, or by flicking the tube.
Spin down briefly in a microfuge.
In the 0.2 ml thin-walled PCR tube containing your cfDNA, mix the following:
Reagent | Volume |
---|---|
cfDNA from the previous step | 46 µl |
NEBNext FFPE DNA Repair Buffer v2 | 7 µl |
NEBNext FFPE DNA Repair Mix v2 | 2 µl |
Total | 55 µl |
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Using a thermal cycler with a heated lid set to 50°C, incubate the reaction at 37°C for 15 minutes and hold at 4°C.
Remove the reaction from the thermal cycler and place the tube on ice.
Keeping the tube on ice, add 2 µl of NEBNext Thermolabile Proteinase K directly to the repaired reaction mixture.
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
Using a thermal cycler with a heated lid set to 75°C, incubate at 37°C for 15 minutes and 65°C for 5 minutes, then hold at 4°C.
Remove the reaction from the thermal cycler and place the tube on ice.
Keeping the tube on ice, add 3 µl of NEBNext Ultra II End Prep Enzyme Mix directly to the reaction mixture for a total volume of 60 µl.
Mix by pipetting 10 times, followed by spinning down quickly to collect all liquid from the sides of the tube.
Using a thermal cycler with a heated lid set to 75°C, incubate at 20°C for 30 minutes and 65°C for 30 minutes, then hold at 4°C.
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Add 180 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
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Prepare 1 ml of fresh 80% ethanol in nuclease-free water.
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Keep the tube on the magnet and wash the beads with 300 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
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溶åºæ¶²ãç¡è²éæã«ãªããŸã§ãå°ãªããšã1åéãã°ãããäžã§ããŒãºããã¬ããåããŸãã
61ÎŒlã®æº¶åºæ¶²ãé€å»ããæž æœãª1.5mlãšããã³ãã«ãDNA LoBindãã¥ãŒãã«ä¿æããŸãã
CHECKPOINT
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Note: You should expect to recover approximately 75% of your input mass. For example, from 30 ng of cfDNA, a yield of approximately 20â25 ng is expected.
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Take forward the repaired and end-prepped cfDNA into the adapter ligation step. However, at this point it is also possible to store the sample at 4°C overnight.
5. Adapter ligation and clean-up
ææ
- Ligation Adapter (LA)
- Ligation Buffer (LNB)
- Short Fragment Buffer (SFB)
- AMPure XP Beads (AXP)
- Elution Buffer (EB)
æ¶èå
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- NEBNext Quick Ligation Module (NEB, E6056)
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit⢠Assay Tubes (Invitrogen, Q32856)
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- ãã°ãããã©ãã¯
- å°åé å¿æ©
- ãã«ããã¯ã¹ãããµãŒ
- P1000 ããããåã³ããã
- P100 ãããããšããã
- P20 ãããããšããã
- P10 ãããããšããã
- Qubitèå å 床èšïŒãŸãã¯QCãã§ãã¯ã®ããã®åçåïŒ
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æšå¥šã®ä»ç€Ÿè£œãªã¬ãŒãŒïŒligaseïŒã«ã¯å°çšã®ãããã¡ãŒãä»å±ããŠããŸãããLigation Sequencing Kitã«ä»å±ã®Ligation Buffer (LNB)ã䜿çšããæ¹ããLigation Adapter (LA)ã®ã©ã€ã²ãŒã·ã§ã³å¹çãé«ããªããŸãã
Spin down the Ligation Adapter (LA) and Quick T4 Ligase, and place on ice.
ã©ã€ã²ãŒã·ã§ã³ãããã¡ãŒ(LNB)ã宀枩ã§è解ããã¹ãã³ããŠã³ããŠããããã£ã³ã°ã§æ··åããŸããç²æ§ãé«ãçºããã®ç·©è¡æ¶²ããã«ããã¯ã¹ããã®ã¯å¹æçã§ã¯ãªãã§ãã解åããŠæ··ããããããã«æ°·ã®äžã«çœ®ããŠãã ããã
溶åºãããã¡ãŒïŒEBïŒã宀枩ã§è解ãããã«ããã¯ã¹ã§æ··åããŸãããã®åŸãã¹ãã³ããŠã³ããŠæ°·ã®äžã«çœ®ããŸãã
Thaw the 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:
Between each addition, pipette mix 10-20 times.
Reagent | Volume |
---|---|
DNA sample from the previous step | 60 µl |
Ligation Adapter (LA) | 5 µl |
Ligation Buffer (LNB) | 25 µl |
NEB Quick T4 DNA Ligase | 10 µl |
Total | 100 µl |
åå¿æ¶²ãå®å šã«æ··åããããã«ããã£ãããšããããã£ã³ã°ãçæéã¹ãã³ããŠã³ããŠäžããã
åå¿æ¶²ã宀枩ã§10åéã€ã³ãã¥ããŒãããŸãã
AMPure XP ããŒãºïŒAXPïŒããã«ããã¯ã¹ã§æžæ¿ããŸãã
Add 120 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.
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ãµã³ãã«ãã¹ãã³ããŠã³ãããã°ãããäžã§ãã¬ããåããŸãããã¥ãŒãããã°ãããã®äžã«çœ®ããç¡è²éæã«ãªã£ããäžæž ãããããã§åãé€ããŸãã
Wash the beads by adding 250 ÎŒl of Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
Note: Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.
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ã¹ãã³ããŠã³ãããã¥ãŒãããã°ãããã®äžã«æ»ããŸããæ®ã£ãäžæž ãããããã§åãé€ããŸãããã¬ããã30 ç§éçšä¹ŸãããŸãã ãã ãã ãã¬ããã«ã²ã³ãå ¥ããŸã§ã¯ä¹Ÿç¥ãããªãã§ãã ããã
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 room temperature.
溶åºæ¶²ãç¡è²éæã«ãªããŸã§ãå°ãªããšã1åéãã°ãããäžã§ããŒãºããã¬ããåããŸãã
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
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Note: You should expect to recover approximately 35-50% of input mass. For example, from 30 ng cfDNA a yield of 10-15 ng of adapter ligated library is expected.
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調補ãããã©ã€ãã©ãªãŒã¯ããããŒã»ã«ãžã®ããŒãã«äœ¿çšãããŸããã©ã€ãã©ãªãŒã¯ãããŒãã®æºåãã§ãããŸã§æ°·äžããŸãã¯4âã§ä¿åããŠäžããã
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çæéã®ä¿åãç¹°ãè¿ã䜿çšããå Žåã¯__ïŒäŸããããŒã»ã«ããŠãªãã·ã¥ããŠå床ããŒãããå ŽåïŒã¯ãã©ã€ãã©ãªãŒãEppendorf DNA LoBindãã¥ãŒãã«å ¥ãã__4âã§ä¿å ããããšããå§ãããŸãã __3ãæ以äžã®é·æä¿åã®å Žåã¯ã____ã©ã€ãã©ãªãŒãEppendorf DNA LoBindãã¥ãŒãã« -80 ° Cã§ä¿å ããããšããå§ãããŸãã
6. Priming and loading the PromethION Flow Cell
ææ
- Sequencing Buffer (SB)
- Library Beads (LIB)
- Flow Cell Tether (FCT)
- Flow Cell Flush (FCF)
æ¶èå
- PromethION Flow Cell
- 1.5 ml Eppendorf DNA LoBind tubes
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- PromethION 2 Solo device
- PromethION sequencing device
- PromethION Flow Cell Light Shield
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
éèŠ
This method is only compatible with R10.4.1 flow cells (FLO-PRO114M).
Thaw the Sequencing Buffer (SB), Library Beads (LIB), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.
ãããŒã»ã«ãã©ã€ãã³ã°ããã¯ã¹ã調補ããã«ã¯ããããŒã»ã«ãã¶ãŒïŒFCTïŒãšãããŒã»ã«ãã©ãã·ã¥ïŒFCFïŒã以äžã®æ瀺éãã«æ··åããŠãã ããããã®åŸã«ã宀枩ã§ãã«ããã¯ã¹ããŠæ··åããŠãã ããã
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ã·ã³ã°ã«ãŠãŒã¹ãã¥ãŒãã®å Žå: 30ÎŒlã®Flow Cell TetherïŒFCTïŒãFlow Cell FlushïŒFCFïŒãã¥ãŒãã«çŽæ¥å ããŠãã ããã
ããã«ãã©ãŒãããã®å Žå: ãããŒã»ã«ã®æ°ã«é©ãããã¥ãŒãã«ã以äžã®è©Šè¬ãå ¥ããŠãã ããs:
è©Šè¬ | ãããŒã»ã«ãããã®å®¹é |
---|---|
Flow Cell Flush (FCF) | 1,170 µl |
Flow Cell Tether (FCT) | 30 µl |
åèš | 1,200 µl |
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å·èµåº«ãããããŒã»ã«ãåãåºããåŸã«ãããŒã»ã«ã宀枩ã«æ»ããŸã§20ååŸ ã£ãŠããPromethIONã«å·®ã蟌ãã§ãã ããã湿床ã®é«ãç°å¢ã§ã¯ãããŒã»ã«ã«çµé²ãçããããšããããŸãããããŒã»ã«ã®äžé¢ãšäžé¢ã«ããéè²ã®ã³ãã¯ã¿ãŒãã³ã«çµé²ããªãããç¹æ€ããçµé²ã確èªãããå Žåã¯ãªã³ãããªãŒã®ãŠã§ãããã£ãã·ã¥ã§æãåã£ãŠãã ããããããŒã»ã«äžé¢ã«ããŒããããïŒé»ããããïŒãããããšã確èªããŠãã ããã
PromethION 2 Soloã®å ŽåããããŒã»ã«ã¯ä»¥äžã®ããã«ã»ããããŸã
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PromethION 24/48 ã®å Žåã¯ããããŒã»ã«ããããã³ã°ããŒãã«ã»ããããŸã
- ãããŒã»ã«ãšã³ãã¯ã¿ãŒãæ°Žå¹³ããã³åçŽã«äžŠã¹ãŠãããæå®ã®äœçœ®ã«ã¹ã ãŒãºã«æ¿å ¥ããŠãã ããã
- ãããŒã»ã«ããã£ãããšæŒãäžããã©ããããã¿åããã«ãããšé³ãããŠæå®ã®äœçœ®ã«åãŸãããšã確èªããŸãã
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ãããŒã»ã«ã誀ã£ãè§åºŠã§æ¿å ¥ãããšãPromethIONã®ãã³ãæå·ããã·ãŒã±ã³ã¹çµæã«åœ±é¿ãåãŒãå¯èœæ§ããããŸããPromethIONã®ãã³ãæå·ããŠããå Žåã¯ãsupport@nanoporetech.com ãŸã§ãé£çµ¡ãã ããã
ã€ã³ã¬ããããŒãã«ããŒãæèšåãã«ã¹ã©ã€ããããŠéããŸãã
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ãããŒã»ã«ãããããã¡ãŒãåŒãäžããéã«ã¯æ³šæããŠãã ããã20ïœ30ÎŒl以äžã¯é€å»ããããã¢ã®ã¢ã¬ã€å šäœãåžžã«ãããã¡ãŒã§èŠãããŠããããšã確èªããŠäžãããã¢ã¬ã€ã«æ°æ³¡ãå ¥ããšããã¢ã«äžå¯éçãªãã¡ãŒãžãäžããå¯èœæ§ããããŸãã
ã€ã³ã¬ããããŒããéããåŸãå°éãã€åŒãæ»ããŠæ°æ³¡ãåãé€ããŸãïŒ
- P1000ãããããããã200µlã«ã»ããããŸãã
- ããããã€ã³ã¬ããããŒãã«æ¿å ¥ããŸãã
- ãã€ã€ã«ã220ïœ230µlã瀺ããŸã§ããŸãã¯ãããããããã«å°éã®ãããã¡ ãŒãå ¥ãã®ã確èªã§ãããŸã§ããã€ãŒã«ãåããŸãã
æ°æ³¡ãå ¥ããªãããã«ã500 µl ã®ãã©ã€ãã³ã°ããã¯ã¹ãã€ã³ã¬ããããŒããããããŒã»ã«ã«æ³šå ¥ããïŒåéåŸ ã¡ãŸãããã®éã«ããããã³ãŒã«ã®æ¬¡ã®ã¹ãããã§ã©ã€ãã©ãªãŒãããŒãããæºåãããŠãã ããã
Library Beads(LIB)ã®æ¶²ãããããã£ã³ã°ããããšã§ååã«æ··åããŠäžããã
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Library BeadsïŒLIBïŒãã¥ãŒãã«ã¯ããŒãºã®æžæ¿æ¶²ãå ¥ã£ãŠããŸãããããã®ããŒãºã¯ããã«æ²æ®¿ããã®ã§ã䜿çšçŽåã«æ··åããããšãéèŠã§ãã
ã»ãšãã©ã®ã·ãŒã±ã³ã¹å®éšã«ã¯Library Beads ïŒLIBïŒã®äœ¿çšãæšå¥šããŸããããããããç²æ§ã®é«ãã©ã€ãã©ãªãŒã«ã¯Library SolutionïŒLISïŒã䜿ã£ãŠãã ããã
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:
Reagent | Volume per flow cell |
---|---|
Sequencing Buffer (SB) | 100 µl |
Library Beads (LIB) thoroughly mixed before use | 68 µl |
DNA library | 32 µl |
Total | 200 µl |
Note: The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.
500ÎŒlã®ãã©ã€ãã³ã°ããã¯ã¹ãã€ã³ã¬ããããŒãã«ãã£ãããšæ³šå ¥ãããããŒã»ã«ã®ãã©ã€ãã³ã°ãå®äºããŸãã
調補ããã©ã€ãã©ãªãŒã¯ãããŒãããçŽåã«ããããã£ã³ã°æ··åããŠäžããã
P1000ããããã䜿çšããŠãã€ã³ã¬ããããŒãã«200µlã®ã©ã€ãã©ãªãŒãæ³šå ¥ããŸãã
ã€ã³ã¬ããããŒããå¯éããããã«ãã«ããéããŸãã
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- ã©ã€ãã·ãŒã«ããšã€ã³ã¬ããããŒãããããŒã»ã«ã®ã€ã³ã¬ããããŒãã«ããŒã«åãããŸããã©ã€ãã·ãŒã«ãã®åçžããããŒã»ã«IDã®äžã«äœçœ®ããããã«ããŸãã
- ã©ã€ãã·ãŒã«ããã€ã³ã¬ããããŒãã«ããŒã®åšå²ã«ãã£ãããšæŒãä»ããŸããã€ã³ã¬ããããŒãã¯ãªãããã€ã³ã¬ããããŒãã«ããŒã®äžã«ã«ãããšã¯ãŸãããã«ãªã£ãŠããŸãã
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MinKNOWã§ã·ãŒã±ã³ã¹ã©ã³ãéå§ããæºåãã§ããããPromethIONã®èãéããŠãã ããã
ãããŒã»ã«ãPromethIONã«ããŒãããåŸãå®éšãéå§ããåã«æäœ10åéåŸ ã¡ãŸãããã®åŸ ã¡æéãããããšã§ãããã·ãŒã±ã³ã¹åºåãåäžããŸãã
7. Data acquisition and basecalling
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Ensure you are using the most recent version of MinKNOW.
We recommend updating MinKNOW to the latest version prior to starting a sequencing run for the best sequencing results.
For more information on updating MinKNOW, please refer to our MinKNOW protocol.
How to start sequencing
Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling.
Please ensure MinKNOW is installed on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.
Please ensure when setting up a sequencing run you are using the recommendations outlined below. All other parameters can be left to their default settings.
MinKNOW can be used and set up to sequence in multiple ways:
- On a computer either direcly or remotely connected to a sequencing device.
- Directly on a PromethION 24/48 sequencing device.
For more information on using MinKNOW on a sequencing device, please see the device user manuals:
Open the MinKNOW software using the desktop shortcut and log into the MinKNOW software using your Community credentials.
Click on your connected device.
Set up a sequencing run by clicking Start sequencing.
Type in the experiment name, select the flow cell postition and enter sample ID. Choose FLO-PRO114M flow cell type from the drop-down menu.
Click Continue to kit selection.
Select the Ligation Sequencing Kit V14 (SQK-LSK114).
An expansion kit does not need to be selected.
Click Continue to Run Options to continue.
Set the run options to a 72 hour run length and 20 bp minimum read length.
Click Continue to basecalling to continue.
Set up basecalling using the following parameters:
- Ensure the basecalling is switched ON.
- Next to "Models", click Edit options and choose High accuracy basecaller (HAC) from the drop-down menu.
- Ensure barcoding is OFF.
Click Continue to output and continue.
Keep the output format and filtering options to their default settings.
Click Continue to final review to continue.
Click Start to start sequencing.
You will be automatically navigated to the Sequencing Overview page to monitor the sequencing run.
Data analysis after sequencing
After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.
After sequencing and basecalling, the data can be analysed, as outlined in the Downstream analysis section.
8. Downstream analysis
Bioinformatics analysis
If basecalling is not performed during live sequencing, raw sequencing data (.POD5 format) can be processed post-sequencing.
This can be achieved using the tool Dorado, which enables basecalling and subsequent alignment to a reference genome.
Dorado can also detect modified bases by using the modified-bases option (e.g. --modified-bases 5mCG_5hmCG
). This will integrate methylation tags directly into the aligned BAM file. We also recommend applying a minimum QScore cutoff (--min-Qscore <min_QScore>
), which serves as a quality control measure to ensure only high-quality reads are used in downstream processes.
1. The command below demonstrates how to initiate basecalling with Dorado, followed by sorting, and indexing the output using Samtools. Please see the Dorado documentation here for further details.
Dorado basecaller <model> <input_POD5> --reference <REF> --min-qscore <min_QScore>
| samtools sort -o <OUTPUT_BAM> - && samtools index <OUTPUT_BAM>
For example to SUP basecall with 5mCG and 5hmCG detected in CpG context, and with a QScore filter of 10 we can use:
Dorado basecaller sup --modified-bases 5mCG_5hmCG input.pod5 --reference ref.fasta --min-qscore 10
| samtools sort -o output.bam > - && samtools index output.bam
2. It is also recommended to remove reads that have a poor alignment score i.e. 10. This can be achieved as follows:
samtools view -q <min_map_q> -bh -o <OUTPUT_BAM> <INPUT_BAM> && samtools index -@ <threads> <OUTPUT_BAM>
3. The output from Dorado basecaller can be demultiplexed into per-barcode BAMs using Dorado demux. E.g.
Dorado demux --output-dir <output-dir> --no-classify <input-bam>
4. You may optionally omit methylation information from read ends usingâ¯modkitâ¯adjust-mods or modkit tools with --edge-filter
option. This may help increase methylation call precision, as the very end of reads, approximately 27 bases, may suffer from loss in methylated bases due to the chemistry used to repair ends in library preparation (see our know-how document for further details).
modkit adjust-mods --edge-filter 0 27 <IN_BAM> <OUTPUT_BAM>
The modified .bam file can be used with external tools that use a .bam file as input for further data analysis and exploration.
Post-basecalling analysis
There are several options for further analysing your basecalled data:
1. EPI2ME workflows
For in-depth data analysis, Oxford Nanopore Technologies offers a range of bioinformatics tutorials and workflows available in EPI2ME. The platform provides a vehicle where workflows deposited in GitHub by our Research and Applications teams can be showcased with descriptive texts, functional bioinformatics code and example data.
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, please refer to the resource centre and search for bioinformatics tools for your application. 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.
9. ãããŒã»ã«ã®åå©çšãšè¿åŽ
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- Flow Cell Wash Kit (EXP-WSH004)
ã·ãŒã¯ãšã³ã¹å®éšçµäºåŸããããŒã»ã«ãåå©çšããå Žåã¯ãFlow Cell Wash Kitã®ãããã³ãŒã«ã«åŸããæŽæµãããããŒã»ã«ã2ïœ8âã§ä¿ç®¡ããŠãã ããã
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