- Materials
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- (FOR EXTRACTION) ≥3.5 ml blood in EDTA K2 vacuum tube or ≥1ml plasma, per sample
- (FOR LIBRARY PREPARATION) ≥6 ng of recovered human cfDNA per sample
- Native Barcoding Kit 24 V14 (SQK-NBD114.24)
- Consumables
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- PromethION Flow Cell
- QIAamp MinElute ccfDNA Midi Kit (QIAGEN, 55284)
- Agencourt AMPure XP beads (Beckman Coulter™, 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)
- NEB Blunt/TA Ligase Master Mix (NEB, M0367)
- NEBNext Quick Ligation Module (NEB, E6056)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Isopropanol
- Freshly prepared 80% ethanol in nuclease-free water
- 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 thin-walled PCR tubes
- Equipment
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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
- Hula mixer (gentle rotator mixer)
- Magnetic rack for 15 ml tubes
- Magnetic rack, suitable for 1.5 ml Eppendorf tubes
- Thermomixer, or other shaker for microcentrifuge tubes, with capacity to heat at 56°C
- Microfuge
- Vortex mixer
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Qubit fluorometer (or equivalent for QC check)
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This protocol has been developed to process and sequence 12 samples. The following inputs are required:
Input requirements per sample for the extraction method:
- ≥3.5 ml of fresh blood in EDTA K2 vacuum tube or ≥1ml plasma, per sample
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:
- Use ≥6 ng of recovered cfDNA, per sample as input. Inputs should be normalised so the same mass of sample is used in each reaction.
Note: This method has been developed for multiplexing 12 samples. We do not recommend deviating from the outlined method.
- ≥3.5 ml of fresh blood in EDTA K2 vacuum tube or ≥1ml plasma, per sample
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Input DNA
How to QC your input DNA
It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.
For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.
Chemical contaminants
Depending on how the DNA is extracted from the raw 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 of the Community.
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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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Check your flow cell
We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within three months of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.
Flow cell Minimum number of active pores covered by warranty Flongle Flow Cell 50 MinION/GridION Flow Cell 800 PromethION Flow Cell 5000 -
Native Barcoding Kit 24 V14 (SQK-NBD114.24) contents
Note: We are in the process of reformatting our kits with single-use tubes into a bottle format and reducing the concentration of EDTA.
Single-use tubes format with higher EDTA concentration:
Higher concentration of EDTA with a clear cap.Bottle format with reduced EDTA concentration:
Reduced concentration of EDTA with a blue cap.Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.
Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.