TITLE: Tn5-Duplex-Sequencing (Tn5-Duplex-Seq) for low-input single-molecule variant detection
AUTHORS: Diane Shao, Nazia Hilal, Sangita Choudhury

[DESCRIPTION]
DNA mutations are the inevitable consequences of errors that arise during replication-repair of DNA damage as well as aging and disease progression. Because of their random and infrequent occurrence, quantification, and characterization of DNA mutations in the genome of somatic cells have been difficult. These mutations in DNA drive genetic diversity, alter gene function, define evolutionary trajectories, and provide targets for precision medicine and diagnostics. It is crucial to detect mutations across a wide range of abundance, i.e., variant allele frequency (VAF). Detecting low-abundance mutations (e.g., <0.1–1% VAF or in individual cells) is important for understanding human embryonic development, somatic mosaicism, and clonal hematopoiesis and uncovering pathogenic variants. Altogether somatic mutations provide important and unique insights into the biology of complex diseases. To decipher the causal inference, we must build robust genetic maps of somatic evolution in health and disease. The recent advent of duplex consensus sequencing has heralded a new generation of accuracy. However, multiple techniques focus on targeted areas of the genome (Twin Strand Biosciences) or are limited to restriction sites (Nanoseq), limiting their application to comprehensive somatic variant characterization. Furthermore, fragmentation of the genome and standard A-tailing and ligation creates errors (BotseqS, CODEC). Ligation of duplex strands for efficient sequencing has proven promising, though in practice requires complex molecular structures (Pro-Seq, CODEC) which have been observed to frequently result in incorrectly paired duplexes (CODEC). To enable comprehensive variant detection by next-generation DNA sequencing, we propose an innovative, accessible, and highly accurate Tn5 transposase-based duplex-sequencing technology (Tn5-duplex-seq) where complementary strands of DNA could be labeled at the molecular level in a single-tube reaction; thus, identifying single nucleotide variants (SNVs) from single-molecules
of DNA regardless of starting from single cells or pooled cell/DNA input. The conceptual basis of the protocol comes from META-CS (Xing et al.2021), a Tn5 based aproach optimized for single-cell whole genome amplification. We find that modifications of this approach to include flexible input and the sequencing strategy to optimize cost per variant detection enables great flexibility for all low-input applications.
Tn5-duplex-seq approach offers several benefits over other duplex approaches including. 

(1) preservation of original template molecules by utilizing 16 unique sequences (Compared to the loss of 50% of
molecules due to intramolecular symmetry during TN5-based Nextera library preparation) 
(2) accuracy by eliminating the requirement for A-tailing 
(3) efficiency of duplex capture through specifying input 
(4) accessibility by using standard reagents and oligonucleotide preparations 
(5) distinction between double-stranded SNVs and single-stranded lesions. 
 
Our method enables library preparation for short-read sequencing. Downstream analysis enables accurate and high-throughput SNV/indel and copy number analysis.

[GUIDELINES]
Optimization of proteinase K concentration for Step 1:
As different cell types may vary in the degree of chromatin condensation and material, we recommend titration of proteinase K at 0.5X, 1X, 5X, 10X our recommended concentration. The final library yield will indicate the optimal degree of digestion. In particular, this current protocol is optimized for nuclei or extracted DNA, and whole cells will likely require a higher concentration.

Expected yield and curve prior to selection for 50 cells (Note: can skip this visualization step for low yields):
ZymoClean 200ul binding buffer + 50ul reaction. Elute in 15ul TE. Run on HS Bioanalyzer.
Yield 4-8ng/ul.

Sequencing suggestion: 
Ideally, part A, and B should all be sequenced separately to avoid the fragment length bias of the Illumina sequencer and to recover the most from the single-cell genome.

For cost consideration, part A alone can be sequenced on  NovaSeq X Plus 10B (2x150bp) , with a 20% PhiX spike-in, which
should be sufficient for determining the single-cell mutation rate.

[STEPS]
SECTION: RECIPE FOR MAKING IN-HOUSE REAGENTS
2. 2X Single Cell Lysis Buffer 
   40 millimolar (mM) 
   40 millimolar (mM) 
    0.3 % volume 
   
 

12X quenching solution
  600 millimolar (mM) 
  90 millimolar (mM)    0.02 % volume 

 
ADP1 and ADP2 Mix
 Reconstitute the 16 ADP1 and 16 ADP2 primers separately in low TE and store in aliquots at -80 °C until ready for use.
Make an equimolar mix of the 16 ADP1 and ADP2 primers to make the ADP1 and ADP2 mix respectively.

 


SECTION: PROTOCOL MATERIALS
1.     
    
    
    
                    
  100 millimolar (mM)   
  10 millimolar (mM)   
   20 mg/mL 
    
    
    
        
    
    
    
    
  
  

SECTION: TRANSPOSOME LOADING
3. Transposon Annealing
Reconstitute 16 META-CS oligos and 1 reverse oligo to 100 micromolar (µM) in Annealing Buffer (40 millimolar (mM)Tris-HCl (pH 8), 50 millimolar (mM) NaCl)
Combine 1:1 of a singular META-CS oligo with the reverse oligo (there should be 16 separate reactions to put on the thermocycler). Mix up the reaction, spin it down briefly, and run the
thermocycler using the conditions below:

 





Transposon Assembly.
Combine all 16 reactions into one tube and aliquot for storage at -80 °C.
Take 10 μLof this aliquot and combine it with 10μL of unloaded Transposome 
Incubate at 23 °C for30 min
Add 10 μL of 100% glycerol.
Aliquot and store at -80 °C.
 

SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
4. Sorting and lysing cells 2 μL 
Prepare nuclei for sorting.
Sort cells directly into 2 μL of 1X cell lysis buffer 
Run the thermocycler using the conditions below 65 °C

30 °C for 60 min
    55 °C for 10 min
4 °C hold
The plate can be stored after lysis.
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
5. Tn5 tagmentation 8 μL 
Add 8 μL transposition mix (total 10 μL reaction). Vortex, spin down.
5 μLDiagenode 2X Tagmentation buffer
1 μL diluted Tn5 per optimized dilution instructions above
2 μL H2O
Incubate in thermocycler using the conditions below 65 °C

55 °C for 15 min
4 °C hold
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
6. Quenching 2 μL 
Prepare 6X Stop Mix and add 2 μL Mix per tube. Spin down, vortex, and spin down.
Incubate in thermocycler using the conditions below 65 °C

37 °C for 30 min
55 °Cfor 10 min
4 °C hold
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
7.  First Strand tagging 13 μL 
Add 13 μL Strand Tagging Mix 1. Vortex and spin down.
5 μL Q5 Reaction Buffer
5 μL μL Q5 High GC Enhancer
0.6 μL 100 millimolar (mM) MgCl2
0.5 μL 10 millimolar (mM) dNTP mix
0.25 μL BSA 20mg/ml
0.25 μL Q5 polymerase 
0.85 μL100 micromolar (µM) ADP1 primer mix
0.55 μL H2O
Incubate in thermocycler using the conditions below 105 °C 

72 °C for 3 min
  98 °C for 30 s
62 °C for 5 min
72 °C for 1 min
4 °C hold
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
8. Stop reaction 1 μL 
Add 1 μL Thermolabile ExoI per tube. Try to touch the minimum of the solution surface. Spin down first, then plate mix, and spin down again.

37 °C for 15 min
65 °C for 5 min
4 °C hold  75 °C
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
9. Second Strand tagging 4 μL 
Add 4 μL Strand Tagging 2 Mix (total 30 μL). Vortex and spin down.
1 μL Q5 Reaction Buffer
1 μL Q5 High GC Enhancer
0.95 μL 100 micromolar (µM) ADP2 primer mix
0.1 μL 10 millimolar (mM) each dNTP mix
0.1 μL Q5 polymerase
0.85 μL H2O
Incubate in thermocycler using the conditions below 105 °C. 

98 °Cfor 30 s
62 °C for 5 min
72 °C for 1 min
4 °C hold
SECTION: TN5-DUPLEX LIBRARY  PROCEDURE
10. Stop reaction1 μL  
Add 1 μL Thermolabile ExoI per tube. Try to touch the minimum of the solution surface. Spin down first, then plate mix, and spin down again. 

 37 °C for 15 min
 65 °C for5 min  
 4 °Chold 75 °C

SECTION: PURIFICATION AND ZYMO CLEAN
12. Zymo clean
Utilize the Zymo DNA Clean & Concentrator Kit  with associated protocol (abbreviated version below).
 For microbulk samples, use 4:1 DNA binding buffer to sample (200 μL buffer to 50 μLreaction). For single cell samples, pool desired samples first, then measure the total pooled volume and use 4:1 DNA binding buffer to sample volume. For single cell samples, pool 5 cells per spin column. For 50 cell samples, use 1 spin column per sample. 
 Add it to the spin column. The maximum volume that the spin column can hold is 800 μL so pooled samples should have to be run through the same column sequentially until all of the liquid has been run through, discarding flowthrough each time.
Spin for 30 s at maximum speed on the tabletop centrifuge >10,000xg at RT
 Add 200 μL wash buffer (with ethanol added) and centrifuge column
 Repeat wash again
 Add 42 μL x0.1 TE to elute and wait 4 min at room temperature
 Spin for 30 s at maximum speed on the tabletop centrifuge >10,000xg at RT
2.  Run 2 μL on High Sensitivity D5000 TapeStation chip. 


SECTION: Appendix-1
14.  SI Appendix  Table S1: Oligonucleotide sequences of META-CS transposon DNA and primers.

All oligos are HPLC purified 
 
  
 
 
11. Library prep14 μL 
 Make PCR Mix (per cell):
 5 μL NEB Universal Primer (NEB E7335S, E7500S, E7710S, E7730S)
 4 μL Q5 Reaction Buffer 
4 μLQ5 High GC Enhancer
0.4 μL 10 millimolar (mM) each dNTP mix
0.4 μL water (H2O)
 0.2 μL Q5 polymerase *add last
2.Add 5 μL NEB Index Primer    per tube, avoiding touching the liquid.
3.Add 14 μL PCR Mix per tube, avoiding touching the liquid. Vortex and spin down.
4. Incubate in thermocycler using the conditions below 

98 °C for 20 s
12 cycles of 98 °C for 10 s, 72 °C for 2 min
72 °C for 5 min
4 °C hold
 

SECTION: PURIFICATION AND ZYMO CLEAN
13. Size Selection (AMPure) 40 μL 
    Add 22 μL(0.55X) resuspended AMPure XP beads to 40 μL DNA library.
Vortex and spin down. Label the tube as “A”. Incubate for 5 min at RT.
    Place tube A on a magnetic stand for 5 min. Carefully transfer the supernatant to a
new tube. Label the new tube as “B”. 
     Size select tube “A” (0.55x AMPure XP beads):
a.    Add 200 μL of 80% freshly prepared ethanol to all tubes while in the magnetic stand, then carefully remove and discard the supernatant.
b.     repeat the ethanol wash step one more time. 
c.     Let air dry on magnetic stand for 1 min at RT. 
d.     Remove the tubes from the magnetic stand. Elute DNA from beads with 12 μL 0.1X TE (for
single cell pools) or 18 μL 0.1X TE (for 50 cell pools). Vortex and gently spin down, incubate for 3 min at RT.
e.     Place the tubes on the magnetic stand for 3 min. Transfer 18 μL of supernatant to a clean tube.
    Size select tube "B" (0.8x AMPure XP beads):
a.    Add 8 μL AMPure XP beads to DNA solution in tube B (0.15X). Vortex
and gently spin down. Incubate for 5 min at RT.
b.    Place tube B on the magnetic stand for 5 min. Remove supernatant from tube B.  
                                              
i.     IMPORTANT: save the supernatant in case AmPure did not work! (the DNA will still be in the supernatant)
c.     Add 200 μL of 80% freshly prepared ethanol to all tubes while in the magnetic stand, then carefully remove and discard the supernatant.
d.     repeat the ethanol wash step one more time. 
e.     Let air dry on magnetic stand for 1 min at RT. 
f.      Remove the tubes from the magnetic stand. Elute DNA from beads with 12 μL 0.1X TE (for
single cell pools) or 18 μL 0.1X TE (for 50 cell pools). Vortex and gently spin down, incubate for 3 min at RT.
g.     Place the tubes on the magnetic stand for 3 min. Transfer 18 μL of supernatant to a clean tube.
    Run 2 μLon High Sensitivity D5000 TapeStation chip. (Run1 μL of sample and 1 of 0.1x TE instead of 2 μL of sample if you need to preserve sample). (Expected concentration for 5 single cell pools is: 300-1000 pg/μL. Expected concentration for 50 cell pools is: 2000-15000 pg/μL .
    Proceed to DNA quantification with final product and dilute accordingly for sequencing.
Run TapeStation, need at least 5 nanomolar (nM) concentration for sequencing. *Fraction B
yields best sequencing results.