High-throughput nanopore sequencing of cell-free DNA

Billy T Lau

Published: 2023-04-04 DOI: 10.17504/protocols.io.4r3l27rjxg1y/v1

Abstract

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.

Steps

Multiplexed cfDNA library preparation (barcode ligation)

1.

25µL of each Sample (usually this is half of the extracted volume, in case of reaction failure) is diluted with 25µL of water in a PCR strip tube or microtiter plate.

2.

A master mix of end-repair and a-tailing mix according to vendor instructions. Briefly, 7µL of end-repair buffer and 3µL of end-repair enzyme is combined together to create a master mix. Use 10-20% overage.

3.

Add 10µL of master mix to each cfDNA sample to obtain 60µL total volume. Pipet mix.

4.

Incubate at 20°C followed by 65°C .

5.

Add 5µL water, 30µL ligation buffer from , and 5µL of a sample barcode from to each well. Add 10µL ligation enzyme from . Mix thoroughly.

6.

Place samples in a thermocycler and incubate at 20°C followed by 4°C .

Ligation cleanup

7.

Add 88µL of to each well and mix thoroughly. You can use any off-brand beads. We use . Incubate for 0h 5m 0s .

8.

Pool all samples together into a 50ml centrifuge tube. Magnetize using a

Equipment

ValueLabel
Dynamag-50 Separation MagnetNAME
MagnetTYPE
Thermo Fisher ScientificBRAND
12302DSKU
https://www.thermofisher.com/us/en/home.htmlLINK

for at least 0h 20m 0s . This may take much longer depending on the number of samples. The supernatant should be completely clear with no haziness.

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.

10.

Pulse centrifuge the 50ml tube and magnetize. Remove any residual ethanol. Repeat this step three times.

11.

Elute in 600µL 10mM Tris-HCl pH 8.0 buffer. Close the centrifuge tube tightly and vortex to resuspend the beads. Incubate for 0h 5m 0s for full elution. Magnetize the beads and remove the elution buffer. Store in a fresh 1.5ml microcentrifuge tube.

12.

Perform a second bead cleanup by adding 900µL Ampure XP beads to the pooled samples. Incubate for 0h 5m 0s .

13.

Place on a

Equipment

ValueLabel
DynaMag-2NAME
MagnetTYPE
InvitrogenBRAND
12321DSKU

and magnetize for at least 0h 5m 0s . Remove the supernatant and wash twice with 80% ethanol. Remove any residual ethanol by pulse centrifugation and magnetizing twice.

14.

Elute in 50µL 10mM Tris-HCl pH 8.0 buffer. Incubate for at least 0h 5m 0s for full elution. Magnetize and remove elution buffer and place in new PCR strip tube.

Nanopore adapter ligation

15.

Add 7µL of end repair buffer and 3µL end repair enzyme to the pooled sample. Incubate at 20°C followed by 65°C .

16.

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

17.

Incubate at room temperature for at least 1h 30m 0s .

18.

Add 88µL of to the ligation reaction. Incubate for 0h 5m 0s . Magnetize the beads and discard the supernatant.

19.

Add 200µL 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.

20.

Resuspend in 25µL of EB buffer from .

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.

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