TITLE: CompDuplex: Accurate detection of somatic mutations by duplex-seq with comprehensive genome coverage
AUTHORS: Muchun Niu, Chenghang Chuck Zong

[DESCRIPTION]
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]
SECTION: Custom Tn5 transposase assembly
1.    
Prepare 500 µL of 2X Annealing Buffer.
40 µL   
10 µL   
450 µL    




SECTION: Custom Tn5 transposase assembly
2. Transposon annealing.
Prepare the following mix in a PCR tube.

20 µL  2X Annealing Buffer
10 µL  200 micromolar (µM) 
10 µL  200 micromolar (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 50 micromolar (µM)  .



3. Custom Tn5 assembly.

Prepare the following mix in a 1.5 mL tube. 
20 µL   
20 µL 50 micromolar (µM) 

Gently pipet to mix, and incubate at 23 °C  for 30 min.

Add 20 µL    to the tube, mix well, aliquot, label as “Tn5_BtgZ1”, and store at -20 °C .


SECTION: CompDuplex Procedure
4. 1. Re-purification of genomic DNA

Dilute 20 ng    to 10 µL nucleus-free water in a PCR tube. Add 5 µL    (0.5X) to the tube, mix well, and incubate at Room temperature  for 5 min. 

Put the tube on the magnetic stand. When the aqueous phase becomes transparent, remove the supernatant, wash twice with 120 µL 80 % volume. Air-dry the beads.


None. 


SECTION: CompDuplex Procedure
5. 2. On-bead custom Tn5 tagmentation

Prepare the following tagmentation mix.
2 µL   
0.48 µL 1:30 diluted Tn5_BtgZ1
7.52 µL   


Resuspend the air-dried beads with 10 µL 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 3 µL  of 0.2M EDTA.
Incubate on a thermal cycler at 50 °C for 30 min.

SECTION: CompDuplex Procedure
7. 4. Restriction enzyme digestion

Prepare the following restriction enzyme digestion mix (Total 15 µL ).
7.5 µL Gap filling product
1.5 µL   
0.7 µL   
5.3 µL 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.



SECTION: CompDuplex Procedure
6. 3. Gap filling

Add 3 µL of 0.2M MgCl2 to the tube.
Add 16 µL   to the tube.
Incubate on a thermal cycler at 65 °C for 3 min.
Quench the reaction by adding 2 µL  of 0.5M EDTA.
Perform double size selection with 0.48X/0.75X   , elute in 7.5 µL water.

SECTION: CompDuplex Procedure
8. 5. Restriction enzyme release

Prepare the following restriction enzyme release mix (Total 500 µL ).

15 µL   
12 µL   
10 µL  1M KCl
5 µL   
18 µL 10% NP40
420 µL Water
20 µL 20 mg/mL   


Add 15 µL 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   , elute in 10 µL water.


SECTION: CompDuplex Procedure
9. 6. Y-shape ligation adapter annealing

Prepare the following 15 micromolar (µM).

1 µL  
10.5 µL 100 micromolar (µM) T1_BtgZ1_ME
7.5 µL 100 micromolar (µM) Rev_T2_BtgZ1
31 µL 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 



SECTION: CompDuplex Procedure
10. 7. Y-shape ligation adapter ligation

Prepare the following ligation mix (Total 50 µL ).
10 µL Sample from Step 8
25 µL   
3 µL  15 micromolar (µM)
3 µL   
9 µL Water

Incubate at 25 °C for 30 min.
Purify with 0.8X   , elute in 51 µL water.

SECTION: CompDuplex Procedure
11. 8. Quantification of library complexity

Dilute 1 µL of ligation product with 9 µL 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.

5 µL   
0.25 µL 10 micromolar (µM) T1ME
0.25 µL 10 micromolar (µM) T2ME
3.5 µL water
1 µL 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



SECTION: CompDuplex Procedure
12. 9. Library amplification

We recommend to amplify 14 cycles for a library with 20 million DNA fragments.

25 µL   
2.5 µL 10 micromolar (µM) Illumina Nextera N5XX index primer
2.5 µL 10 micromolar (µM) Illumina Nextera N7XX index primer
20 µL 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   , elute in 20 µL water.