- Materials
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- >15 ml of whole blood
- Ligation Sequencing Kit XL V14 (SQK-LSK114-XL)
- Ultra-Long DNA Sequencing Kit V14 (SQK-ULK114)
- Flow Cell Wash Kit XL (EXP-WSH004-XL)
- Consumables
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- PromethION Flow Cell (FLO-PRO114M)
- Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Monarch® HMW DNA Extraction Kit for Tissue (NEB, T3060)
- Puregene Blood Kit (QIAGEN, 158023)
- T4 DNA Ligase 400,000 U/ml (NEB, M0202S/L)
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L).
Alternatively, you can use the NEBNext® products below:
- NEBNext® Ultra II End Repair / dA-tailing Module (NEB, E7546)
- NEBNext Quick Ligation Module (NEB, E6056)
- NEBNext FFPE DNA Repair Mix (NEB, M6630)
- RBC Lysis Solution (QIAGEN, 158106)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- 5 M NaCl (Sigma, 71386)
- PEG 8000, 50% w/v (Rigaku Reagents, cat # 25322-68-3)
- TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
- 0.5 M EDTA, pH 8 (Thermo Scientific, R1021)
- Percoll, 1.135 g/ml (Cytiva, 17-0891-01)
- (Optional) dimethyl sulfoxide (DMSO) (Sigma-Aldrich, 20-139)
- ECOSURF EH-9 (Dow, 64366-70-7)
- Foetal bovine serum (FBS) (Gibco™, A3840401)
- Glycine (Sigma, 56-40-6)
- Formaldehyde at 36.5% v/v (Sigma, 33220)
- NlaIII restriction enzyme with CutSmart Buffer (NEB, R0125L)
- IGEPAL CA-630 (Sigma, I8896)
- Protease Inhibitor Cocktail (Sigma, P8340)
- Sodium dodecyl sulfate (SDS) at 10% v/v (Sigma, 71736)
- Tween-20 (Thermo Scientific, J20605.AP)
- 1 M Tris-HCl pH 8.0 (Thermo Scientific, 15893661)
- Proteinase K at 20 μg/μl (NEB, P8107S)
- 10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
- Recombinant Albumin at 20 μg/μl (NEB, B9200S)
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Isopropanol, 100% (Fisher, 10723124)
- Chilled phenol:chloroform:isoamyl alcohol in a 25:24:1 ratio, saturated with 10 mM Tris.HCl pH 8.0, 1 mM EDTA (Sigma, P3803-400ML)
- 3 M sodium acetate, pH 5.5 (Invitrogen, AM9740)
- 50 ml centrifuge tubes
- 15 ml Falcon tubes
- 2 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Ziplock bags
- 0.2 µm filter
- Equipment
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- PromethION 24/48 device
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P10 pipette and tips
- P20 pipette and tips
- Wide-bore pipette tips
- Pasteur pipettes
- Thermal cycler or heat block
- Hula mixer (gentle rotator mixer)
- Magnetic rack
- Vortex mixer
- Temperature-controlled centrifuge
- Microfuge
- Ice bucket with ice
- Thermomixer
- Qubit fluorometer (or equivalent for QC check)
- Class I hood with active charcoal filter
- -80°C freezer storage
- Optional equipment
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- Liquid nitrogen and canister
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Approximately 15 ml of whole blood is required as input for the sample preparation steps.
Aliquots of whole blood are prepared for each experiment to isolate the peripheral blood mononuclear cells (PBMCs) for the Pore-C and Ultra-long DNA experiments and to extract gDNA for the Duplex experiment.
The whole blood can be collected in an anticoagulant such as K2-EDTA but we do not recommend mixing with other additives as they may interfere with the Pore-C DNA extraction or the DNA sequencing run.
We recommend different sample preparations due to different input requirements for each experiment. However, users may use other methods they feel are most appropriate but to ensure the individual input requirements are followed. Depending on how DNA is extracted from a sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page.
Input requirements:
- Pore-C experiment: 10 million PBMCs
- Duplex experiment: 1 μg or 100-200 fmol extracted DNA
- Ultra-long DNA experiment: 6 million PBMCs
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We recommend preparing your samples and the custom SPRI bead suspension a day ahead of the experiments to ensure maximum use of time each day.
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Third-party reagents
We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.
For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.
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NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
We recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L). This contains all the NEB reagents needed for use with the Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) during day 1 of the Duplex experiment and day 4 of the Pore-C experiment.
Please note, the Pore-C experiment also requires additional use of the T4 DNA Ligase kit for the proximity ligase reaction during day 2 of the protocol.
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Ligation Sequencing Kit XL V14 (SQK-LSK114-XL) contents
Name Acronym Vial colour Number of vials Fill volume per vial (µl) DNA Control Strand DCS Yellow 1 100 Ligation Adapter LA Green 1 320 Ligation Buffer LNB White 1 1,500 Elution Buffer EB White cap, black strip label 1 10,000 Long Fragment Buffer LFB White cap, orange strip label 2 20,000 Short Fragment Buffer SFB White cap, blue strip label 2 20,000 Library Beads LIB Pink 2 1,800 Library Solution LIS White cap, pink label 2 1,800 Sequencing Buffer SB Red 3 1,700 Flow Cell Flush FCF Clear 4 15,500 Flow Cell Tether FCT Purple 1 1,600 Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.
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Ultra-Long DNA Sequencing Kit (SQK-ULK114) contents
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 XL (EXP-WSH004-XL) contents
Name Acronym Fill volume per vial (µl) Cap colour No. of vials No. of uses Wash Mix WMX 150 Brown 1 48 Wash Diluent DIL 20,000 White cap, brown stripe 1 48 Storage Buffer S 25,000 White cap, yellow stripe 1 48 - 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.