High-throughput nanopore sequencing of cell-free DNA
Billy T Lau
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)
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.
A master mix of end-repair and a-tailing mix 7µL of end-repair buffer and 3µL of end-repair enzyme is combined together to create a master mix. Use 10-20% overage.
Add 10µL of master mix to each cfDNA sample to obtain 60µL total volume. Pipet mix.
Incubate at 20°C followed by 65°C .
Add 5µL water, 30µL ligation buffer from 5µL of a sample barcode from 10µL ligation enzyme from
Place samples in a thermocycler and incubate at 20°C followed by 4°C .
Ligation cleanup
Add 88µL of 0h 5m 0s .
Pool all samples together into a 50ml centrifuge tube. Magnetize using a
Equipment
| Value | Label |
|---|---|
| Dynamag-50 Separation Magnet | NAME |
| Magnet | TYPE |
| Thermo Fisher Scientific | BRAND |
| 12302D | SKU |
| https://www.thermofisher.com/us/en/home.html | LINK |
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.
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.
Pulse centrifuge the 50ml tube and magnetize. Remove any residual ethanol. Repeat this step three times.
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.
Perform a second bead cleanup by adding 900µL Ampure XP beads to the pooled samples. Incubate for 0h 5m 0s .
Place on a
Equipment
| Value | Label |
|---|---|
| DynaMag-2 | NAME |
| Magnet | TYPE |
| Invitrogen | BRAND |
| 12321D | SKU |
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.
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
Add 7µL of end repair buffer and 3µL end repair enzyme to the pooled sample. Incubate at 20°C followed by 65°C .
Add 30µL ligation buffer from 10µL of AMX-F adapter from 10µL ligation enzyme from
Incubate at room temperature for at least 1h 30m 0s .
Add 88µL of 0h 5m 0s . Magnetize the beads and discard the supernatant.
Add 200µL of SFB wash buffer from
Resuspend in 25µL of EB buffer from
Quantify the libraries using
