This is a Legacy document. We recommend using our new updated methods for the best results:

Cell free DNA (cfDNA) repository

• Cell free DNA (cfDNA) info sheet

Extraction only protocols:

• Human blood cell-free DNA (cfDNA) extraction for singleplex sequencing
• Human blood cell-free DNA (cfDNA) extraction for multiplex sequencing

Full method protocols (Sample extraction, library preparation and data processing):

• Ligation sequencing V14 — Human cfDNA singleplex (SQK-LSK114)
• Ligation sequencing V14 — Human cfDNA multiplex (SQK-NBD114.24)

Introduction

This protocol describes 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 was performed using the QIAGEN QIAamp MinElute ccfDNA Midi Kit. The cfDNA libraries were prepared using the Ligation Sequencing Kit (SQK-LSK114) with a modified sequencing protocol, as detailed below in the Method section.

Materials

• 10 ml of human blood in EDTA K2 vacuum tube
• QIAamp MinElute ccfDNA Midi Kit
• Ligation Sequencing Kit V14 (SQK-LSK114)
• AMPure XP Beads (AXP)

Consumables

• NEBNext FFPE DNA Repair v2 Module (NEB, cat # E7360)
• NEBNext® Ultra II End Repair / dA-tailing Module (NEB, cat # E7546)
• Quick T4 DNA ligase, from NEBNext Quick Ligation Module (E6056)
• Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
• 100% Ethanol
• Isopropanol
• Freshly prepared 80% ethanol in nuclease-free water
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Qubit™ Assay Tubes (Invitrogen, Q32856)
• 5 ml DNA LoBind Eppendorf 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
• Microcentrifuge
• Hula mixer, or a gentle rotation mixer
• Magnetic rack for 15 ml tubes
• Magnetic rack for microcentrifuge tubes
• Thermomixer, or other shaker for microcentrifuge tubes, with capacity to heat at 56°C
• Vortex
• P1000 pipette and tips
• P100 pipette and tips
• P10 pipette and tips
• Thermal cycler
• Microfuge
• Hula mixer (gentle rotator mixer)
• Magnetic rack
• Ice bucket with ice
• Qubit fluorometer (or equivalent for QC check)

Method

Extraction

1. Centrifuge the blood in the EDTA K2 vacuum tube at 1900 x g for 10 minutes at 4°C in a swing out rotor centrifuge.
2. Pipette and transfer the supernatant (this is the plasma fraction) to a fresh 5 ml DNA LoBind Eppendorf tube. The volume should be around 3.5–4 ml.
3. Centrifuge the plasma at 16,000 x g for 10 minutes (or 6000 x g for 30 minutes, depending on the spin capacity of the centrifuge) at 4°C, in a fixed angle rotor.
4. Aspirate the supernatant and transfer it to a fresh 15 ml tube.
5. Follow the recommend protocol from steps 1-11 (pages 17-19 of the QIAamp MinElute ccfDNA Handbook.
6. Quantify 1 µl of eluted sample using a Qubit fluorometer.

DNA repair and end-prep protocol

1. Use 15–30 ng of recovered cfDNA as input.
2. In a 0.2 ml thin-walled PCR tube, prepare the following reaction:

Component	Volume
cfDNA (15–30 ng)	46 µl
FFPE DNA Repair Buffer v2	7 µl
NEBNext FFPE DNA Repair Mix v2	2 µl
Total volume	55 µl

3. Mix by pipetting 10 times and briefly spin down.
4. 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.
5. Place the reation mixture on ice.
6. Keeping the tube on ice, add 2 µl of NEBNext Thermolabile Proteinase K directly to the repaired reaction mixture.
7. Mix by pipetting 10 times followed by a quick spin to collect all liquid from the sides of the tube.
8. Place back in thermal cycler, with a heated lid set to 75°C, and incubate at 37°C for 15 minutes and 65°C for 5 minutes, then hold at 4°C.
9. Place the reaction mixture on ice.
10. 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.
11. Mix the reaction by pipetting 10 times and briefly spin down.
Note: It is important to mix the reaction well. The presence of a small amount of bubbles will not interfere with the reaction performance.
12. Using a thermal cycler with a heated lid set to 75°C, incubate the reaction at 37°C for 30 minutes, then 65°C for 30 minutes and hold at 4°C.
13. Resuspend the AMPure XP Beads (AXP) by vortexing.
14. Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
15. Add 180 µl of resuspended AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.
16. Incubate on a Hula mixer (rotator mixer) for 5 minutes at room temperature.
17. Prepare 500 µl of fresh 80% ethanol in nuclease-free water.
18. Spin down the sample and pellet on a magnet until the supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.
19. Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
20. Repeat the previous step.
21. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
22. Remove the tube from the magnetic rack and resuspend the pellet in 61 µl nuclease-free water. Incubate for 2 minutes at room temperature.
23. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
24. Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
25. Quantify 1 µl of eluted sample using a Qubit fluorometer.

END OF STEP: Take forward the repaired and end-prepped DNA into the adapter ligation step. However, at this point it is also possible to store the sample at 4°C overnight.

Modified Ligation Sequencing gDNA protocol

Follow the rest of the protocol from adapter ligation and clean-up, using the following modifications:

1. 'Adapter ligation and clean-up' section, step 9: use 120 µl of resuspended AMPure XP Beads (AXP).
2. In the 'Adapter ligation and clean-up' section, we recommend using Short Fragment Buffer (SFB) in step 12.
3. When setting up the sequencing run, we recommend using Short Fragment Mode (SFM) and setting the minimum read length to 20 bp to avoid losing short fragments. For instructions on setting this up, see the 'Starting a sequencing run' section for your device in the MinKNOW protocol.

Results

• Yield: 15-30 ng per 10 ml of blood (3.5-4 ml of plasma)

Sequencing performance

Libraries were prepared using the Ligation Sequencing kit and sequenced using the modified protocol above.

• Read length profile:

Bioinformatics analysis

1. If not performed during live sequencing, raw sequencing data (i.e. pod5 format) can be basecalled and aligned to a reference using dorado. Methylation tags will be automatically incorporated into the aligned bam output. This can be achieved by selecting the option of modified bases (e.g. --modified-bases 5mCG_5hmCG) and specifying a reference as shown below:
   
   

    dorado basecaller <MODEL> <POD5_FOLDER> --modified-bases 5mCG_5hmCG --reference <REF> > <OUTPUT_BAM> 

2. It is recommended to remove reads that have a poor alignment score i.e. 10.
   
   

    samtools view -q <min_map_q> -bh -o <OUTPUT_BAM> <INPUT_BAM> && samtools index -@ <threads> <OUTPUT_BAM> 

3. You may optionally omit methylation information from read ends using modkit 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 technical reasons (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.

Appendix

If genome alignment was not performed during basecalling, then alignment of the unaligned bam file can be performed by converting the unaligned bam file to fastq, whilst incorporating the methylation tag information in the fastq header. This can be performed using samtools fastq, and the use of the -T "*" option, as shown below. The fastq file (with retained tags) can then be aligned to a reference using minimap2 with -y which copies the input FASTA/Q comments (methylation tags) to bam output and --MD which encodes deletions and mismatches in relation to the reference.

   samtools fastq -T "*" <INPUT_BAM> |
   minimap2 -y --MD -t <threads> -ax map-ont -Y <REF.fasta> - |
   samtools sort -@ <threads> -T <sample_name> -O BAM -o <OUTPUT_BAM> - && 
   samtools index -@ <threads> <OUTPUT_BAM> 

Change log

Version	Change
v5, 15th May 2024	Protocol status has been changed to Legacy
v4, November 2023	Updated protocol to include human plasma. Updated methodology. Addition of bioinformatics analysis instructions
v3, August 2023	Updated URL links
v2, June 2022	Updated the modified ligation sequencing step for 'Adapter ligation and clean-up' section to use 120 µl rather than 300 µl
v1, May 2022	Initial protocol publication