TITLE: SMRT-Tag - Direct transposition of native DNA for sensitive multimodal single-molecule sequencing
AUTHORS: Scott Nanda, Ke Wu, Iryna Irkliyenko, Siva Kasinathan, vijay.ramani

[DESCRIPTION]
Here we describe a protocol for SMRT-Tag: Single-Molecule Real-Time sequencing by Tagmentation - a transposase-mediatedstrategy for producing low-input, multiplexable, PCR-free PacBio sequencing libraries. 

SMRT-Tag requires only 1-10% as much input material as existing PacBio protocols (typically at least 1–5 µg, or 150,000–750,000 human cells) and enables highly-sensitive and simultaneous detection of sequence variation and CpG methylation comparable to the current state-of-the-art.

In brief, high-quality genomic DNA is tagmented using Tn5 transposase loaded with custom hairpin adaptors, gap-repaired to generate closed and circular templates,  exo-digested to remove unrepaired molecules, and then sequenced on the PacBio Sequel II platform. All steps from purified gDNA to sequencing-ready libraries can be performed in one day. We have demonstrated the utility of SMRT-Tag on input as low as 40ng gDNA (~ 7000 cell equivalents) sequenced monoplex. 


 



[GUIDELINES]
Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Arjun S Nanda1,2*, Ke Wu1*, Iryna Irkliyenko1*, Brian Woo2,6, Megan S Ostrowski1, Andrew S Clugston5,6, Leanne C. Sayles5,6, Lingru Xu6, Ansuman T Satpathy4,7,8, Hao G. Nguyen6, E Alejandro Sweet-Cordero5,6, Hani Goodarzi2,6,8,9, Sivakanthan Kasinathan3,4*#, Vijay Ramani1,2,6,9#

1Gladstone Institute for Data Science & Biotechnology, Gladstone Institutes, San Francisco, CA 94158
2Department of Biochemistry & Biophysics, UCSF, San Francisco, CA 94143
3Division of Rheumatology, Department of Pediatrics, Stanford University, Stanford, CA 94305
4Gladstone-UCSF Institute for Genomic Immunology, Gladstone Institutes, San Francisco, CA 94143
5Department of Pediatrics, UCSF, San Francisco, CA 94143
6Helen-Diller Cancer Center, San Francisco, CA 94158
7Department of Pathology, Stanford University, Stanford, CA 94305
8Parker Institute for Cancer Immunotherapy, San Francisco, CA 94129
9Bakar Computational Health Sciences Institute, San Francisco, CA 94143
*These authors contributed equally to this work.
#correspondence to skas@stanford.edu or vijay.ramani@gladstone.ucsf.edu


[STEPS]
SECTION: Annealing SMRT-Tag adaptors
1. Reconstitute HPLC-purified SMRT-Tag adaptors (IDT) to 100 µM in nuclease-free water.
SECTION: Annealing SMRT-Tag adaptors
2. Dilute adaptors to 20 µM in 1x annealing buffer (10mM Tris-HCl pH 7.5 and 100mM NaCl) in PCR tubes.
SECTION: Annealing SMRT-Tag adaptors
3. Place the tubes in a thermocycler at 95°C with a heated lid at 105ºC for 5 min, then remove them from the heat source and slowly cool to room temperature for at least 30 min. 

[NOTE] Annealed adaptors can be stored at -20°C for up to 6 months.
SECTION: Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)
4. Thaw Tn5 enzyme stock (3.9mg/mL) suspended in Storage Buffer (50mM Tris-HCl​ pH 7.5, 800mM NaCl, 0.2mM EDTA, 2mM DTT, 10% glycerol) on ice.

SECTION: Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)
5. Dilute to ~1mg/mL (or 18.9 µM monomer) in Tn5 dilution buffer (50mM Tris-HCl pH 7.5, 200mM NaCl, 0.1mM EDTA, 2mM DTT, and 50% glycerol) and slowly mix 10-15 times with a wide-bore pipette. The mixture is extremely viscous. 

SECTION: Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)
6. Let sit on a rotational mixer at 4ºC for at least 3 hr until the mixture becomes fully homogenized.
SECTION: Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)
7. Mix 1.02x volume of 1mg/mL Tn5 with 1x volume of 20 µM annealed SMRT-Tag adaptors using a wide-bore pipette.

SECTION: Assembling SMRT-Tn5 transposomes (Tn5 loaded with SMRT-Tag adaptors)
8. Incubate the mixture at 23ºC for 55 min with continuous shaking at 350 rpm. After 55 min, place on ice. Loaded SMRT-Tn5 (~9.4µM monomer) can be supplemented with glycerol up to a final concentration of 50% and stored at −20° for up to 6 months. 
SECTION: (Optional) Validation of SMRT-Tn5 loading
9. Add 1-2 µL of each loaded SMRT-Tn5 transposome with 2.5µL of 4x Native Gel Loading Buffer and dilute up to 10 µL with dH2O.

SECTION: (Optional) Validation of SMRT-Tn5 loading
10. Load each prepared sample on a NativePAGE 4-16% Bis-Tris Gel along with GeneRuler Ultra Low Range DNA Ladder, a negative DNA control (Tn5 without SMRT-Tag adaptors), a negative protein control (annealed SMRT-Tag oligos without Tn5), a positive control (Tn5 loaded with any confirmed adaptors i.e Nextera N5/N7 oligos) if available.
SECTION: (Optional) Validation of SMRT-Tn5 loading
11. Add 200mL of cold cathode buffer (1x NativePAGE running buffer and 0.1x NativePAGE cathode additive) to the inner chamber. Add 600mL of cold anode buffer (1x NativePAGE running buffer) to the outer chamber.
SECTION: (Optional) Validation of SMRT-Tn5 loading
12. Run the gel at 150V for 1 hr at 4ºC, followed by 180V for 15 min.
SECTION: (Optional) Validation of SMRT-Tn5 loading
13. Stain the gel with 1x SYBR Gold Solution for ~30 min on a shaker. Visualize on a LiCor or any imaging system to confirm the proper loading of SMRT-Tn5 transposomes. 


 

SECTION: (Optional) Validation of SMRT-Tn5 loading
14. Stain the gel with 1x Coomassie Blue for 1 hr at RT on a shaker, destain for at least 20 min with dH2O, and image using visible light to confirm the presence of protein band.

 

SECTION: (Optional) Validation of SMRT-Tn5 enzymatic activity
15. For each tagmentation reaction, add 2 µL of 5x TAPS buffer (50mM TAPS-NaOH pH 8.5, 25mM MgCl2), 1 µL of 100% DMF, 160ng of genomic DNA (gDNA) as a template, 1 µL of serially diluted SMRT-Tn5 (i.e 1x, 4x, 16x, 64x), and dilute up to 10 µL with dH2O. 
SECTION: (Optional) Validation of SMRT-Tn5 enzymatic activity
16. Incubate reactions (10 µL) at a range of temperatures (i.e. 37°C, 55°C) for a range of incubation periods (i.e 30 min, 60 min). To validate activity, we recommend 37ºC and 55ºC for 30 min.
SECTION: (Optional) Validation of SMRT-Tn5 enzymatic activity
17. Terminate the reactions by adding 2.5 µL of 0.2% SDS and incubate for 5 min at RT. 
SECTION: (Optional) Validation of SMRT-Tn5 enzymatic activity
18. Mix each reaction (12.5 µL) with 2.5 µL of 6x loading dye and load on a 0.5% agarose gel. Use 5µL of NEB 1kb-Extend for the ladder. Run at 60-80V for 2-3 hr.
SECTION: (Optional) Validation of SMRT-Tn5 enzymatic activity
19. Stain the gel with 1x SYBR Gold Solution in TAE buffer for ~30 min on a shaker. Visualize on a LiCor or any imaging system to confirm the gel shift of transposase activity - DNA size distribution post tagmentation varies as a function of SMRT-Tn5 concentration. 

 

SECTION: Quality control of gDNA
20. High Molecular Weight genomic DNA (HMW gDNA) is used as standard input for the SMRT-Tag method. High quality DNA is essential for efficient sequencing.

To reduce gDNA viscosity, incubate at 37ºC for 5-10 min at low agitation speed (300 rpm). Pipette up and down 5–10 times using a P200 wide-bore pipette to ensure any clumps of DNA are dispersed.

[OPTIONAL] Storing gDNA at 4°C for days or weeks also facilitates homogenization and relaxation of HMW gDNA. 
 


SECTION: Quality control of gDNA
21. Once homogenized, measure the concentration and purity of HMW gDNA on a microvolume spectrophotometer (e.g. Nanodrop). Qubit-based concentration values are >25% consistently lower than OD-based estimates.
 


SECTION: Quality control of gDNA
22. Dilute QC-passing HMW gDNA to 50-100 ng/µL with 1x EB (or 10 mM Tris-HCl, pH 8.5) and measure the diluted concentration with Qubit 1x High Sensitivity dsDNA Assay.

[CRITICAL] Do not dilute gDNA in TE buffer since EDTA will decrease the Tn5 efficiency in downstream tagmentation reactions.

[OPTIONAL] Analyze 1µL of diluted HMW gDNA using Genomic DNA Screentape (Agilent) to determine a DNA Integrity Number (DIN). We recommend at minimum that samples have DIN > 9.0.
SECTION: Tagmentation
23. Prepare the Tagmentation Reagent Mix:
 

[CRITICAL] We recommend the following dilution factors when using barcoded SMRT-Tn5 to generate PacBio libraries of 2 different size distributions:
Use a 1:64 (64x) dilution factor if the target library size is <3kb and using Sequel II Binding Kit 2.1.
Use  a 1:128 (1:128x) dilution factor if the target library size is >3kb and using Sequel II Binding Kit 2.0 / 2.2.

SECTION: Tagmentation
24. Incubate the tagmentation reactions at 55ºC for 30 min using a thermocycler or thermomixer and hold at 4ºC.
SECTION: Tagmentation
25. Add 2.5 µL of 0.2% SDS per sample. Pipette slowly to mix and let incubate at room temperature for 5 min. 

[NOTE] Placing the tubes on ice after 5 min can help reduce bubble formation. 
SECTION: Tagmentation
26. Dilute each sample (12.5 µL) to 30 µL with 1x EB and add 2x volume of resuspended, room-temperature SPRI beads to clean up the reactions. Mix well using a P200 with wide-bore tips.
SECTION: Tagmentation
27. Incubate the bead-mixed samples in a thermomixer at 23ºC for 30 min with interval mixing @ 350 rpm (1 min on, 3 min off) to keep the beads resuspended.

[NOTE] Longer binding times noticeably increase the recovery efficiency of tagmented DNA.
SECTION: Tagmentation
28. Spin down quickly. Place on the magnet and allow the solution to clear before carefully withdrawing the supernatant. 
SECTION: Tagmentation
29. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~30 s to 1 min to dry. Do not over-dry the beads - dried beads are light-brown and appear to be fragmented and cracked. 
SECTION: Tagmentation
30. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the tagmented fragments. Once beads are resuspended, mix well.
SECTION: Tagmentation
31. Incubate the samples in a thermomixer at 37ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to maximize the yield of DNA elution. 

[NOTE] Incubation at 37ºC with mild agitation improves recovery. Incubations longer than 15 min have not been tested.
SECTION: Tagmentation
32. Spin samples down quickly. Place on the magnet and allow the solution to clear before carefully transferring the supernatant to a new LoBind tube. 
SECTION: Gap Repair
33. Prepare the Repair Reaction Mix: 
 

SECTION: Gap Repair
34. Incubate the gap-repair reactions at 37ºC for 1 hr in a thermocylcer or thermomixer and hold at 4ºC.

[NOTE] Incubation at 50ºC has been tested to improve gap repair efficiency. 
SECTION: Gap Repair
35. Add 2x volume (~ 40µL) of homogeneous, room-temperature SPRI beads to clean up the reactions. Mix well using a P200 with wide-bore tips.
SECTION: Gap Repair
36. Incubate the bead-mixed samples in a thermomixer at 23ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: Gap Repair
37. Spin down quickly. Place on the magnet and allow the solution to clear before carefully withdrawing the supernatant. 
SECTION: Gap Repair
38. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~30 s to 1 min to dry. Do not over-dry the beads.
SECTION: Gap Repair
39. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute DNA. Once beads are resuspended, mix well.
SECTION: Gap Repair
40. Incubate the samples in a thermomixer at 37ºC for 10 min with interval mixing @350 rpm (1 min on, 3 min off) to maximize the yield of DNA elution. 
SECTION: Gap Repair
41. Spin samples down quickly. Place on the magnet and allow the solution to clear before carefully transferring the supernatant to a new LoBind tube. 
SECTION: Gap Repair
42. [OPTIONAL] QC check - take a 1µL aliquot and determine the gap-repaired sample dsDNA concentration using Qubit 1x High Sensitivity dsDNA Assay. 
SECTION: Exonuclease Digestion
43. Prepare the ExoDigest Reaction Mix.

 

SECTION: Exonuclease Digestion
44. Incubate the exonuclease digestion reactions at 37ºC for 1 hr in a thermocycler or thermomixer, then hold at 4ºC.
SECTION: Exonuclease Digestion
45. Add 2x volume (~30µL) of homogeneous, room-temperature SPRI beads to clean up the reactions. Mix well using a P200 with wide-bore tips.
SECTION: Exonuclease Digestion
46. Incubate the bead-mixed samples in a thermomixer at 23ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: Exonuclease Digestion
47. Spin down quickly. Place on the magnet and allow the solution to clear before carefully withdrawing the supernatant. 
SECTION: Exonuclease Digestion
48. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~30 s to 1 min to dry. Do not over-dry the beads.
SECTION: Exonuclease Digestion
49. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the extracted DNA. Once beads are resuspended, mix well.
SECTION: Exonuclease Digestion
50. Incubate the samples in a thermomixer at 37ºC for 10 min with interval mixing @ 350 rpm (1 min on, 3 min off) to maximize the yield of DNA elution. 
SECTION: Exonuclease Digestion
51. Spin samples down quickly. Place on the magnet and allow the solution to clear before carefully transferring the supernatant to a new LoBind tube. 
SECTION: (Optional) Size Enrichment for HMW libraries
52. [CRITICAL] If intending to load the target library using Sequel II Binding Kit 2.1 (i.e libraries generated using a 64x dilution factor of SMRT-Tn5 in step 23), this step can be skipped. If intending to load with Sequel II Binding Kit 2.2 (using the 128x dilution factor of SMRT-Tn5 in step 23), a size enrichment step is critical for enriching for HMW library >5kb. 

Make a 35% v/v dilution of AMPure PB beads by mixing 1.75 mL of room-temperature, resuspended AMPure PB beads with 3.25 mL of 1X EB. The 35% dilution Ampure PB beads can be stored at 4°C for 30 days.
SECTION: (Optional) Size Enrichment for HMW libraries
53. Dilute the library to 50 µL with 1x EB if the sample volume is <50 µL. Add 3.1x volume of resuspended, room-temperature 35% AMPure PB beads using a wide-bore pipette and mix gently at least 10 times.

[OPTIONAL] If multiplexing libraries, pool all libraries together before size enrichment. 
SECTION: (Optional) Size Enrichment for HMW libraries
54. Incubate the bead-mixed samples in a thermomixer at 23ºC for 30 min with interval mixing @350 rpm (1 min on, 3 min off) to keep the beads resuspended.
SECTION: (Optional) Size Enrichment for HMW libraries
55. Spin down quickly. Place on the magnet and allow the solution to clear before carefully withdrawing the supernatant. 

[NOTE] The remaining bead pellet will be small because the SPRI reagent is diluted. Adjust the pipetting technique accordingly.

[OPTIONAL] Save the supernatant (which contains <5kb library molecules) to sequence with Sequel II Binding Kit 2.1. To recover the <5kb fraction (LMW), add 0.25x regular AMPure PB, bind at RT for 10 min, wash twice with 80% EtOH, and elute in 12 µL of 1x EB following 5 min of incubation at RT.
SECTION: (Optional) Size Enrichment for HMW libraries
56. Wash beads twice with 80% ethanol for 30 sec. Add ethanol to the wall opposite of beads. Withdraw supernatant and after a quick spin, remove the remaining liquid with a P20 pipette. Beads take ~30 s to 1 min to dry. Do not over-dry the beads.
SECTION: (Optional) Size Enrichment for HMW libraries
57. Remove from the magnet stand, and gently resuspend the bead pellet in 12 µL 1x EB (or 10 mM Tris-HCl, pH 8.5) to elute the extracted DNA. Once beads are resuspended, mix well.
SECTION: (Optional) Size Enrichment for HMW libraries
58. Incubate the samples in a thermomixer at 37ºC for 15 min with interval mixing @350 rpm (1 min on, 3 min off) to maximize the yield of DNA elution. 
SECTION: (Optional) Size Enrichment for HMW libraries
59. Spin samples down quickly. Place on the magnet and allow the solution to clear before carefully transferring the supernatant to a new LoBind tube. 
SECTION: Library QC and Sequencing
60. Determine the concentration of SMRT-Tag libraries using a Qubit 1x High Sensitivity dsDNA Assay. 

Determine the library size distribution using Agilent 2100 Bioanalyzer High Sensitivity dsDNA Assay. 

If the sample is precious, library size may be roughly estimated at ~3000bp for SMRT-Tag libraries prepared with a 64x dilution factor, and ~7000bp for SMRT-Tag libraries prepared with 128x dilution factor and >5kb size selection. 

[CRITICAL] Convert the Bioanalyzer trace from its default unit (fluorescence units, FU) to "molarity per length" using the R package "bioanalyzeR". We have found this conversion step is critical to accurately size SMRT-Tag libraries, as is required for the optimal loading on a PacBio Sequel II. 
 


 




SECTION: Library QC and Sequencing
61. Sequence SMRT-Tag libraries using a PacBio Sequel II 8M SMRTcell with either Sequel II Binding Kit 2.1 (e.g. up to 220pM loading concentration with 30h movie time, 2h pre-extension, and 4h immobilization), or Sequel II Binding Kit 2.2 (e.g. up to 120pM loading concentration with 30h movie time, 2h pre-extension, and 4h adaptive loading).

Samples may be sequenced in monoplex or multiplex formats. For sample multiplexing, we recommend pooling libraries to equalize their molar concentration (nM).

[NOTE] For precious libraries, the "before" and "after" loading cleanup Qubit measurements may be skipped, and the entire library loaded on one cell.