CompDuplex: Accurate detection of somatic mutations by duplex-seq with comprehensive genome coverage
Muchun Niu, Chenghang Chuck Zong
Disclaimer
Patent application covering CompDuplex chemistry has been filed by Baylor College of Medicine.
Abstract
Somatic mutations continuously accumulate in the human genome, posing vulnerabilities towards aging and increased risk of various diseases. However, accurate detection of somatic mutations at the whole genome scale is still challenging. By tagging and independently sequencing the two complementary strands of DNA, the recent development of duplex-sequencing methods has greatly improved the detection accuracy, however, the limited genome coverage and the compromised compatibility with existing sequencing platforms have constrained the broad applications of these methods.
To overcome these technical challenges, here we developed a duplex sequencing method with comprehensive genome coverage, which we refer to as CompDuplex-seq. The streamlined chemistry of CompDuplex assay allows efficient generation of libraries readily compatible with standard Illumina 2x150 paired-end sequencing. In addition, we validated the accuracy of somatic mutation calling and comprehensive genome coverage of CompDuplex by profiling a single-cell expanded clone. To summarize, CompDuplex chemistry supports genome-wide coverage while maintaining high accuracy, which we believe will facilitate the whole genome characterization of somatic mosaicism in various biological systems.
Steps
Custom Tn5 transposase assembly
Prepare500µL of 2X Annealing Buffer r.
40µL
10µL
450µL
Transposon annealing.
Prepare the following mix in a PCR tube.
20µL 2X Annealing Buffer
10µL 200micromolar (µM)
10µL 200micromolar (µM)
Oligonucleotides sequences
ME_REV : /5Phos/CTGTCTCTTATACACATCT
T_BtgZ1_ME : ATGTGTGGAGCGATG AGATGTGTATAAGAGACAG
Mix up the reaction, spin down, and perform the following reactions on a thermal cycler.
95°C
65°C
4°C
This results in a 50micromolar (µM) .
Custom Tn5 assembly.
Prepare the following mix in a 1.5 mL tube.
20µL
20µL 50micromolar (µM)
Gently pipet to mix, and incubate at 23°C for 30 min.
Add 20µL -20°C .
CompDuplex Procedure
1. Re-purification of genomic DNA
Dilute 20ng Sample to 10µL nucleus-free water in a PCR tube. Add 5µL Room temperature for 5 min.
Put the tube on the magnetic stand. When the aqueous phase becomes transparent, remove the supernatant, wash twice with 120µL 80% volume. Air-dry the beads.
2. On-bead custom Tn5 tagmentation
Prepare the following tagmentation mix .
2µL
0.48µL 1:30 diluted Tn5_BtgZ1
7.52µL
Resuspend the air-dried beads with 10µL tagmentation mix .
Incubate on a thermal cycler using the following program: 20°C for 5 min, 55°C for 15 min.
Quench the reaction by adding 3µL of 0.2M EDTA.
Incubate on a thermal cycler at 50°C for 30 min.
3. Gap filling
Add 3µL of 0.2M MgCl2to the tube.
Add 16µL
Incubate on a thermal cycler at 65°C for 3 min.
Quench the reaction by adding 2µL of 0.5M EDTA.
Perform double size selection with 0.48X/0.75X 7.5µL water.
4. Restriction enzyme digestion
Prepare the following restriction enzyme digestion mix (Total 15µL ).
7.5µL Gap filling product
1.5µL
0.7µL
5.3µL Water
Incubate on a thermal cycler with the following program: 25°C for 10 min, 37°C for 3 h, 10°C for 12 h.
5. Restriction enzyme release
Prepare the following restriction enzyme release mix (Total 500µL ).
15µL
12µL
10µL 1M KCl
5µL
18µL 10% NP40
420µL Water
20µL 20mg/mL
Add 15µL restriction enzyme release mix to the sample.
Incubate on a thermal cycler with the following program: 50°C for 45 min, 25°C for 2 h.
Purify with 0.85X 10µL water.
6. Y-shape ligation adapter annealing
Prepare the following 15micromolar (µM).
1µL
10.5µL 100micromolar (µM) T1_BtgZ1_ME
7.5µL 100micromolar (µM) Rev_T2_BtgZ1
31µL Water.
Oligonucleotides sequences
T1_BtgZ1_ME : TCGTCGGCAGCGTC AGATGTGTAT
Rev_T2_BtgZ1 : /5Phos/ TCTT ATACACATCT CCGAGCCCACGAGAC
Mix up the reaction, spin down, and perform the following reactions on a thermal cycler.
65°C
20°C
7. Y-shape ligation adapter ligation
Prepare the following ligation mix (Total 50µL ).
10µL Sample from Step 8
25µL
3µL 15micromolar (µM)
3µL
9µL Water
Incubate at 25°C for 30 min.
Purify with 0.8X 51µL water.
8. Quantification of library complexity
Dilute 1µL of ligation product with 9µL water. Use the 1:10 diluted product to quantify library complexity using qPCR. Any Illumina Nextera libraries with known concentration can be used as a standard. An example qPCR procedure is shown below.
5µL
0.25µL 10micromolar (µM) T1ME
0.25µL 10micromolar (µM) T2ME
3.5µL water
1µL template
Oligonucleotides sequences
T1ME : TCGTCGGCAGCGTC AGATGTGTATAAGAGACAG
T2ME : GTCTCGTGGGCTCGG AGATGTGTATAAGAGACAG
94°C 2 min
30 cycles of
`94°C` 20s
`68°C` 20s
`72°C` 1 min, fluorescence scanning
Melting curve
9. Library amplification
We recommend to amplify 14 cycles for a library with 20 million DNA fragments.
25µL
2.5µL 10micromolar (µM) Illumina Nextera N5XX index primer
2.5µL 10micromolar (µM) Illumina Nextera N7XX index primer
20µL ligation product and water
Oligonucleotides sequences
Illumina Nextera N5XX index primer : AATGATACGGCGACCACCGAGATCTACAC NNNNNNNN TCGTCGGCAGCGTC
Illumina Nextera N7XX index primer : CAAGCAGAAGACGGCATACGAGAT NNNNNNNN AGATGTGTATAAGAGACAG
PCR cycles:
98°C 30s
14 cycles of
`98°C` 10s
`63°C` 30s
`72°C` 1 min
72°C 3 min
Purify with 0.8X 20µL water.
