Apr 22, 2025
MAGNET-seq
This protocol is a draft, published without a DOI.
- Dongin Lee1,
- Taehoon Kim1,
- Duhee Bang1
- 1Department of Chemistry, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Yonsei synbio
Protocol Citation: Dongin Lee, Taehoon Kim, Duhee Bang 2025. MAGNET-seq. protocols.io https://protocols.io/view/magnet-seq-eb8qbarvx
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 21, 2025
Last Modified: April 22, 2025
Protocol Integer ID: 131056
Keywords: targeted sequencing, cancer genotyping, multiplex PCR, hybrid capture, cancer mutations
Funders Acknowledgements:
National Research Foundation of Korea (NRF)
Grant ID: RS-2024-00338316
Abstract
MAGNET-seq integrates multiplex PCR with hybrid capture for optimal enrichment of tens of target variants.
It achieves increased on-target ratios across targets without the need for extensive primer design or balancing.
Guidelines
1. MAGNET-seq was validated using 10 ng of input DNA. A higher input amount is recommended to improve detection performance.
2. For data processing, refer to the SPIDER-seq pipeline (Lim et al., bioRxiv, https://doi.org/10.1101/2024.11.26.625438).
Materials
Seraseq ctDNA MRD Panel Mix (AF 0%, 0.005%, 0.05%, 0.5%)
Seraseq ctDNA CompleteTM Mutation Mix (AF 0.1%, 1%)
Phusion U Multiplex PCR Master Mix (Thermo Scientific)
KAPA HiFi HotStart PCR Kit (KAPA Biosystems)
Oligo Clean & Concentrator (Zymo Research)
Celemics Target Enrichment Kit (Celemics)
Target primers
Illumina P5 index primer
Illumina P7 index primer
P5 binding primer
P7 binding primer
Nuclease-free water
100% ethanol
AMPure XP beads (Beckman Coulter)
Dynabeads MyOne Streptavidin T1 beads (Invitrogen)
D1000 ScreenTape (Agilent)
Before start
Prepare a pooled primer mix (total 10 μM) by combining equal volumes of each target-specific primer at the same concentration.
Targeted multiplex PCR
Targeted multiplex PCR
Prepare a 40 μL PCR reaction mix as described in the reagent table below. Aliquot 5 μL of the master mix into each of 8 tubes for target amplification.
| A | B | |
| Phusion U Multiplex PCR Master Mix | 20 L | |
| Nuclease-free water | (16 - X) L | |
| 10M Target primer mix | 4 L | |
| Sample | X L | |
| Total | 40 L |
Perform PCR according to the thermal cycling conditions outlined in the PCR program table.
| A | B | C | |
| 98°C | 30 s | ||
| 98°C | 10 s | 10 cycles | |
| 60°C | 30 s | ||
| 72°C | 30 s | ||
| 72°C | 10 min | ||
| 4°C | hold |
Index PCR
Index PCR
Prepare a 50 μl PCR reaction using the components listed in the reagent table below. Add the index PCR reagents directly to the targeted PCR amplicons without purification.
| A | B | |
| Phusion U Multiplex PCR Master Mix | 25 μL | |
| Nuclease-free water | 15 μL | |
| 10μM Illumina P5 index primer | 2.5 μL | |
| 10μM Illumina P7 index primer | 2.5 μL | |
| Targeted PCR amplicon | 5 μL | |
| Total | 50 μL |
Perform PCR according to the thermal cycling conditions outlined in the PCR program table.
| A | B | C | |
| 98°C | 30 s | ||
| 98°C | 10 s | 10 cycles | |
| 60°C | 30 s | ||
| 72°C | 30 s | ||
| 72°C | 10 min | ||
| 4°C | hold |
DNA purification
DNA purification
Pool all eight index PCR amplicons (total 400 μL) into a 1.5 mL DNA LoBind tube.
Add 480 μL of Ampure XP beads (1.2X volume ratio) and mix thoroughly.
Incubate for 5 minutes at room temperature.
Place the tube on a magnetic stand for 2 minutes, or until the solution clears.
Remove the supernatant.
Wash the beads twice with 700 μL 80% ethanol.
Air-dry the beads for 2 minutes at room temperature.
Elute the purified DNA in 100 μL of nuclease-free water.
DNA concentration
DNA concentration
Pool multiple samples to a final volume of 50 μl.
Concentrate the DNA using the Oligo Clean & Concentrator‱ kit (Zymo Research), following the manufacturer’s instructions.
Elute the final product in 7 μl of nuclease-free water.
Hybrid capture
Hybrid capture
Perform hybrid capture using the Celemics Target Enrichment Kit (Celemics) with the whole exome sequencing panel. Incubate the hybridization reaction for 8 hours following the manufacturer's protocol.
Amplification of the captured library
Amplification of the captured library
Prepare a 100 μL PCR reaction using the components listed in the reagent table below.
| A | B | |
| 2X KAPA master mix | 50 μL | |
| Nuclease-free water | 10 μL | |
| 10μM P5 binding primer | 5 μL | |
| 10μM P7 binding primer | 5 μL | |
| on-beads captured library | 30 μL | |
| Total | 100 μL |
Perform PCR according to the thermal cycling conditions outlined in the PCR program table.
| A | B | C | |
| 98°C | 45 s | ||
| 98°C | 15 s | 20 cycles | |
| 60°C | 30 s | ||
| 72°C | 1 min | ||
| 72°C | 10 min | ||
| 4°C | hold |
DNA purification
DNA purification
Add 120 μL of Ampure XP beads (1.2X volume ratio) and mix thoroughly.
Incubate for 5 minutes at room temperature.
Place the tube on a magnetic stand for 2 minutes, or until the solution clears.
Remove the supernatant.
Wash the beads twice with 200 μL 80% ethanol.
Air-dry the beads for 2 minutes at room temperature.
Elute the purified DNA in 60 μL of nuclease-free water.
Library quantification
Library quantification
Library quantification was performed using D1000 ScreenTape (Agilent).
Protocol references