TITLE: High-throughput nanopore sequencing of cell-free DNA
AUTHORS: Billy T Lau

[DESCRIPTION]
https://www.biorxiv.org/content/10.1101/2022.06.22.497080v1

Epigenetic characterization of cell-free DNA (cfDNA) is an emerging approach for detecting and characterizing diseases such as cancer. We developed a strategy using nanopore-based single-molecule sequencing to measure cfDNA methylomes. This approach generated up to 200 million reads for a single cfDNA sample from cancer patients, an order of magnitude improvement over existing nanopore sequencing methods. We developed a single-molecule classifier to determine whether individual reads originated from a tumor or immune cells. Leveraging methylomes of matched tumors and immune cells, we characterized cfDNA methylomes of cancer patients for longitudinal monitoring during treatment.

[GUIDELINES]
This protocol assumes that you are sequencing multiple cfDNA samples on the Oxford Nanopore Technologies' PromethION system.

This protocol also assumes you have an existing method of extracting cfDNA from plasma.

[STEPS]
SECTION: Multiplexed cfDNA library preparation (barcode ligation)
1. 25 µL of each   (usually this is half of the extracted volume, in case of reaction failure) is diluted with 25 µL of water in a PCR strip tube or microtiter plate.

SECTION: Multiplexed cfDNA library preparation (barcode ligation)
2. A master mix of end-repair and a-tailing mix   according to vendor instructions. Briefly, 7 µL of end-repair buffer and 3 µL of end-repair enzyme is combined together to create a master mix. Use 10-20% overage.

SECTION: Ligation cleanup
7. Add 88 µL of   to each well and mix thoroughly. You can use any off-brand beads. We use   . Incubate for 5 min .

SECTION: Multiplexed cfDNA library preparation (barcode ligation)
3. Add 10 µL of master mix to each cfDNA sample to obtain 60 µL total volume. Pipet mix.

SECTION: Multiplexed cfDNA library preparation (barcode ligation)
4. Incubate at 20 °C followed by 65 °C . 

SECTION: Multiplexed cfDNA library preparation (barcode ligation)
5. Add 5 µL water, 30 µL ligation buffer from   , and 5 µL of a sample barcode from   to each well. Add 10 µL ligation enzyme from   . Mix thoroughly.

SECTION: Multiplexed cfDNA library preparation (barcode ligation)
6. Place samples in a thermocycler and incubate at 20 °C  followed by 4 °C .

SECTION: Ligation cleanup
8. Pool all samples together into a 50ml centrifuge tube. Magnetize using a 
 
for at least 20 min . This may take much longer depending on the number of samples. The supernatant should be completely clear with no haziness.

SECTION: Ligation cleanup
9. Aspirate out the supernatant with a 50ml serological pipet, taking care to not disturb the beads. Wash the beads twice with 80% ethanol using a serological pipet by slowly pipetting the ethanol down the side of the centrifuge tube without disturbing the beads.
SECTION: Ligation cleanup
10. Pulse centrifuge the 50ml tube and magnetize. Remove any residual ethanol. Repeat this step three times.
SECTION: Ligation cleanup
11. Elute in 600 µL 10mM Tris-HCl pH 8.0 buffer. Close the centrifuge tube tightly and vortex to resuspend the beads. Incubate for 5 min for full elution. Magnetize the beads and remove the elution buffer. Store in a fresh 1.5ml microcentrifuge tube.

SECTION: Nanopore adapter ligation
15. Add 7 µL  of end repair buffer and 3 µL end repair enzyme to the pooled sample. Incubate at 20 °C followed by 65 °C .  

SECTION: Ligation cleanup
12. Perform a second bead cleanup by adding 900 µL Ampure XP beads to the pooled samples. Incubate for 5 min .

SECTION: Ligation cleanup
13. Place on a 
 
and magnetize for at least 5 min . Remove the supernatant and wash twice with 80% ethanol. Remove any residual ethanol by pulse centrifugation and magnetizing twice.

SECTION: Ligation cleanup
14. Elute in 50 µL 10mM Tris-HCl pH 8.0 buffer. Incubate for at least 5 min for full elution. Magnetize and remove elution buffer and place in new PCR strip tube.

SECTION: Nanopore adapter ligation
16. Add 30 µL ligation buffer from   , and 10 µL of AMX-F adapter from   . Add 10 µL ligation enzyme from   . Mix thoroughly.

SECTION: Nanopore adapter ligation
17. Incubate at room temperature for at least 90 min .

SECTION: Nanopore adapter ligation
18. Add 88 µL of   to the ligation reaction. Incubate for 5 min . Magnetize the beads and discard the supernatant.
SECTION: Nanopore adapter ligation
19. Add 200 µL of SFB wash buffer from   . DO NOT USE ETHANOL!!! Remove the tube from the magnet, cap it, and flick it gently with a pencil to resuspend the beads. Pulse centrifuge, magnetize, and repeat this step one more time.

SECTION: Nanopore adapter ligation
20. Resuspend in 25 µL of EB buffer from   .

SECTION: Nanopore adapter ligation
21. Quantify the libraries using   with an approximate size of 330bp (you can check the size range with    but it's not super critical). Load 150fmol of library per PromethION flow cell using standard loading protocols.