Apr 04, 2023
High-throughput nanopore sequencing of cell-free DNA
- 1Stanford University
Protocol Citation: Billy T Lau 2023. High-throughput nanopore sequencing of cell-free DNA. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27rjxg1y/v1
Manuscript citation:
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 04, 2023
Last Modified: April 04, 2023
Protocol Integer ID: 79967
Funders Acknowledgement:
NCI
Grant ID: R33CA247700
NHGRI
Grant ID: R35HG011292
Clayville Foundation
Abstract
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.
Protocol materials
KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505
In 5 steps
Agencourt AmPure XP beadsCatalog #A63880
In 2 steps
Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01
Step 7
Native Barcoding Expansion 96Oxford Nanopore TechnologiesCatalog #EXP-NBD196
Step 5
Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110
In 3 steps
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Step 21
E-Gel™ EX Agarose Gels 2%Catalog #G401002
Step 21
Multiplexed cfDNA library preparation (barcode ligation)
Multiplexed cfDNA library preparation (barcode ligation)
25 µL of each Sample (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.
A master mix of end-repair and a-tailing mix KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505 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.
Add 10 µL of master mix to each cfDNA sample to obtain 60 µL total volume. Pipet mix.
Incubate at 20 °C 30 minutes followed by 65 °C 30 minutes .
Add 5 µL water, 30 µL ligation buffer from KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505 , and 5 µL of a sample barcode from Native Barcoding Expansion 96Oxford Nanopore TechnologiesCatalog #EXP-NBD196 to each well. Add 10 µL ligation enzyme from KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505 . Mix thoroughly.
Place samples in a thermocycler and incubate at 20 °C 4.5 hours followed by 4 °C overnight .
Ligation cleanup
Ligation cleanup
30m
30m
Add 88 µL of Agencourt AmPure XP beadsContributed by usersCatalog #A63880 to each well and mix thoroughly. You can use any off-brand beads. We use Mag-Bind® TotalPure NGSOmega BiotekCatalog #M1378-01 . Incubate for 00:05:00 .
5m
Pool all samples together into a 50ml centrifuge tube. Magnetize using a
Equipment
Dynamag-50 Separation Magnet
NAME
Magnet
TYPE
Thermo Fisher Scientific
BRAND
12302D
SKU
for at least 00:20:00 . This may take much longer depending on the number of samples. The supernatant should be completely clear with no haziness.
20m
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 600 µL 10mM Tris-HCl pH 8.0 buffer. Close the centrifuge tube tightly and vortex to resuspend the beads. Incubate for 00:05:00 for full elution. Magnetize the beads and remove the elution buffer. Store in a fresh 1.5ml microcentrifuge tube.
5m
Perform a second bead cleanup by adding 900 µL Ampure XP beads to the pooled samples. Incubate for 00:05:00 .
5m
Place on a
Equipment
DynaMag-2
NAME
Magnet
TYPE
Invitrogen
BRAND
12321D
SKU
LINK
and magnetize for at least 00:05:00 . Remove the supernatant and wash twice with 80% ethanol. Remove any residual ethanol by pulse centrifugation and magnetizing twice.
5m
Elute in 50 µL 10mM Tris-HCl pH 8.0 buffer. Incubate for at least 00:05:00 for full elution. Magnetize and remove elution buffer and place in new PCR strip tube.
5m
Nanopore adapter ligation
Nanopore adapter ligation
1h 35m
1h 35m
Add 7 µL of end repair buffer and 3 µL end repair enzyme to the pooled sample. Incubate at 20 °C 30 minutes followed by 65 °C 30 minutes .
Add 30 µL ligation buffer from KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505 , and 10 µL of AMX-F adapter from Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110 . Add 10 µL ligation enzyme from KAPA HyperPrep Kit (PCR-free)RocheCatalog #KK8505 . Mix thoroughly.
Incubate at room temperature for at least 01:30:00 .
1h 30m
Add 88 µL of Agencourt AmPure XP beadsContributed by usersCatalog #A63880 to the ligation reaction. Incubate for 00:05:00 . Magnetize the beads and discard the supernatant.
5m
Add 200 µL of SFB wash buffer from Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110 . 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.
Resuspend in 25 µL of EB buffer from Oxford Nanopore Ligation Sequencing KitOxford Nanopore TechnologiesCatalog #SQK-LSK110 .
Quantify the libraries using Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854 with an approximate size of 330bp (you can check the size range with E-Gel™ EX Agarose Gels 2%Catalog #G401002 but it's not super critical). Load 150fmol of library per PromethION flow cell using standard loading protocols.
Protocol references