- Materials
-
- ≥3.5 ml fresh human blood in EDTA K2 vacuum tube or ≥1ml plasma, per sample
- Consumables
-
- QIAamp MinElute ccfDNA Midi Kit (QIAGEN, 55284)
- Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Isopropanol
- Freshly prepared 80% ethanol in nuclease-free water
- 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
- Equipment
-
- Centrifuge with capacity for 5 ml and 15 ml tubes, and a swing out and fixed angle rotors
- Microfuge
- Hula mixer (gentle rotator mixer)
- Magnetic rack for 15 ml tubes
- Magnetic rack
- Vortex mixer
- P1000 pipette and tips
- P100 pipette and tips
- Thermal cycler
- Ice bucket with ice
- Qubit fluorometer (or equivalent for QC check)
- Optional equipment
-
- Agilent Femto Pulse System (or equivalent for read length QC)
-
Optimised extraction: Human blood cell-free DNA (cfDNA) extraction for multiplex 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 multiplex sequencing.
These instructions describe a method to extract cell-free DNA (cfDNA) from 12 human blood samples collected in EDTA K2 vacuum tubes (step 1), or human plasma (step 3). 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.
-
Optional actionAlternatively, if you have previously extracted and stored your cfDNA sample(s), this can be used directly in the Library preparation section of this protocol.
-
Preparation of plasma from fresh blood
-
Centrifuge ≥3.5 ml of fresh blood (overnight chilled delivery) in the EDTA K2 vacuum tube at 1900 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.
We recommend a minimum plasma volume of 1 ml is used, although volumes up to 4 ml have been validated.
-
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.
-
Aspirate the supernatant and transfer it to a fresh 15 ml tube.
-
Purification of cfDNA from 1–5 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 (96–100%) 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 1 ml plasma (µl) Volume for 2 ml plasma (µl) Volume for 3 ml plasma (µl) Volume for 4 ml plasma (µl) Volume for 5 ml plasma (µl) Plasma 1,000 2,000 3,000 4,000 5,000 Magnetic Bead Suspension 30 60 90 120 150 Proteinase K 55 110 165 220 275 Bead Binding Buffer 150 300 450 600 750 Total volume 1,235 2,470 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 supernatant–Buffer 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 3.5–4 ml of plasma, you can expect a yield of between 15–30 ng cfDNA.
-
Optional actionWe 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.