Ligation sequencing DNA V14 (SQK-LSK114) library preparation and sequencing on MinION

This SOP was derived from ONT GDE_9161_v114_revAB (26 Aug 2025)

Reagents & consumables

• ONT SQK‑LSK114 kit (LA, LNB, LIB, optional LIS, EB, LFB, SFB, SB, FCF, FCT; AXP included with kit).
• NEBNext Companion Module v2 for ONT ligation (NEB E7672S/L): FFPE DNA Repair Mix + FFPE DNA Repair Buffer v2, Ultra II End‑Prep Enzyme Mix, and Salt‑T4 DNA Ligase (M0467). v2 is recommended for improved dA‑tailing/ligation and cost; the earlier E7180 module is compatible.
• Qubit dsDNA HS Assay Kit, Qubit assay tubes.
• Nuclease‑free water, fresh 80% ethanol.
• BSA 50 mg/mL (for priming mix; improves output on R10.4.1).
• 1.5 mL Eppendorf DNA LoBind tubes; 0.2 mL thin‑walled PCR tubes; standard pipette tips.

Equipment

MinION/GridION device + light shield, Hula mixer (gentle rotator), magnetic rack (1.5 mL compatible), microfuge, vortex, thermal cycler, Qubit fluorometer, calibrated P1000/P200/P100/P20/P10/P2 pipettes, timer, ice bucket.

Input DNA requirements & starting amounts

• Use high‑molecular‑weight DNA of suitable purity and length.

• Starting input by fragment length (table on p. 7):

  • Very short (<1 kb): 200 fmol
  • Short (1–10 kb): 100–200 fmol
  • Long (>10 kb): 1 µg.

• Purity guidance (troubleshooting p. 31): 260/280 ≥ 1.8 and 260/230 ≈ 2.0–2.2; contaminants impair ligation/sequencing.

Flow cell check (within 12 weeks of purchase for MinION/GridION)

• Run Flow Cell Check in MinKNOW before library prep; warranty pore minima (table p. 8): Flongle ≥ 50; MinION/GridION ≥ 800; PromethION ≥ 5000. Report within 2 days if below.

DNA repair & end‑prep (~35 min)

Prepare reagents (p. 10–11):

• Thaw on ice; do not vortex FFPE DNA Repair Mix or Ultra II End‑Prep Enzyme Mix. Vortex FFPE Repair Buffer v2; if precipitate appears, bring to RT and mix to dissolve. Spin down before first use each day.

Set up DNA (p. 11):

1. In a LoBind tube, place 1 µg (or 100–200 fmol) DNA; bring to 47 µL with nuclease‑free water. Mix and quick spin. (Optional: include 1 µL DCS for troubleshooting.)

2. In a 0.2 mL PCR tube, assemble 60 µL end‑prep (mix 10–20× after each addition):

   • DNA: 47 µL
   • DNA CS: 1 µL (optional; replace with sample if omitted)
   • FFPE DNA Repair Buffer v2: 7 µL
   • FFPE DNA Repair Mix: 2 µL
   • Ultra II End‑Prep Enzyme Mix: 3 µL.

3. Incubate 20 °C 5 min → 65 °C 5 min, then cool to 4–20 °C (or place on ice).

Bead clean‑up (p. 12): 4. Vortex AMPure XP (AXP) to resuspend. Transfer reaction to a fresh LoBind tube. 5. Add 60 µL AXP; mix by flicking. Hula mixer 5 min RT. 6. Place on magnet; remove supernatant when clear. 7. Wash twice with 200 µL freshly prepared 80% ethanol without disturbing pellet. 8. Spin, return to magnet, remove residual ethanol. Air‑dry ~30 s (do not overdry). 9. Elute by removing from magnet and adding 61 µL nuclease‑free water; incubate 2 min RT. 10. Magnet ≥ 1 min, transfer 61 µL eluate to new LoBind tube. 11. Qubit quantify 1 µL; proceed to ligation (or 4 °C overnight).

Common pitfall: Using <70% ethanol in washes can cause DNA loss at end‑prep (troubleshooting p. 32).

Adapter ligation & clean‑up (~20 min)

Notes (p. 14): Use Salt‑T4 DNA Ligase (M0467); older Quick T4 is compatible but less efficient. Use ONT LNB, not third‑party ligase buffers, for best ligation of LA.

1. Spin down LA and Salt‑T4 Ligase; keep on ice. Thaw LNB at RT, mix by pipetting (viscous; vortex ineffective), then place on ice. Thaw EB; vortex; spin; put on ice. Thaw LFB or SFB; vortex and keep at RT.

2. In a LoBind tube, assemble ligation mix (order matters; mix 10–20× after each addition, p. 15):

   • DNA: 60 µL
   • LA: 5 µL
   • LNB: 25 µL
   • Salt‑T4 DNA Ligase: 10 µL → Total 100 µL. Incubate 10 min RT.

3. Vortex AXP; add 40 µL to ligation; mix by flicking. Hula 5 min RT. Magnet; remove supernatant when clear.

4. Wash ×2 with 250 µL LFB (to enrich >3 kb) or 250 µL SFB (retain all sizes). Spin, magnet, remove residual liquid; air‑dry ~30 s (do not crack pellet).

5. Elute with 15 µL EB; spin and incubate 10 min RT (37 °C improves recovery for HMW DNA). Magnet ≥ 1 min; transfer 15 µL eluate (final DNA library). Qubit 1 µL.

6. Prepare final library in 12 µL EB at the appropriate loading amount (table on p. 16):

   • Very short (<1 kb): 100 fmol
   • Short (1–10 kb): 35–50 fmol
   • Long (>10 kb): 300 ng If below these yields, load the entire library. (Mass‑to‑fmol calculators such as NEB’s can help.) Store on ice/4 °C until loading. Storage: 4 °C short‑term/re‑loading; –80 °C long‑term (>3 months) — see box p. 16–17.

Critical pitfalls (troubleshooting p. 36): • Using ethanol instead of LFB/SFB after ligation denatures motor protein → pore occupancy ~0. • Ligation failure or missing tether can also drive pore occupancy near 0.

Prime and load the flow cell (~10 min)

Prepare priming mix with BSA (p. 18–19): For improved output on R10.4.1, add BSA to 0.2 mg/mL final. Per flow cell (table on p. 19):

• FCF 1,170 µL + BSA (50 mg/mL) 5 µL + FCT 30 µL → 1,205 µL total. Mix by pipetting at RT.

1. Insert flow cell; press down on priming port cover for thermal/electrical contact (p. 19–20). Optionally run Flow Cell Check now if not done. Open priming port.

2. Draw back 20–30 µL via priming port to remove bubbles: set P1000 to 200 µL, insert tip, dial to 220–230 µL until buffer enters tip. Ensure continuous buffer across sensor array (p. 21). Do not remove >30 µL; never expose array to air.

3. Load 800 µL priming mix via priming port slowly; wait 5 min (p. 22). While waiting, resuspend LIB thoroughly (beads settle rapidly; LIS can be used for viscous libraries).

4. Prepare loading mix (p. 23): in a new LoBind tube per flow cell:

   • SB 37.5 µL + LIB 25.5 µL (or LIS 25.5 µL) + DNA library 12 µL → 75 µL total. Mix gently just before loading.

5. Lift SpotON cover; add 200 µL priming mix again via priming port (not SpotON) to complete priming (p. 23–24).

6. Load 75 µL of prepared library dropwise into SpotON; allow each drop to be drawn in before the next (p. 24–25). Close SpotON and priming port (p. 25–26).

7. Fit the light shield immediately after loading (align against the clip; it sits around SpotON; note that it is not secured—handle carefully) (p. 26–27). Close device lid and proceed to run setup.

Note for Mk1D (p. 28): Lid may sit with a small gap around sides when a flow cell is inserted—normal and does not affect performance.

Start sequencing & basecalling (MinKNOW)

1. In MinKNOW, click Start sequencing; enter experiment name, flow‑cell position, sample ID.
2. Select Kit: Ligation Sequencing Kit V14 (SQK‑LSK114).
3. Configure run and output parameters (defaults are acceptable). If real‑time basecalling is off, basecall post‑run in MinKNOW.
4. Click Start. (Run setup steps summarized from pp. 28–29.)

Flow cell reuse or return

• To reuse, wash promptly using Flow Cell Wash Kit (EXP‑WSH004) and store washed flow cell at +2 – +8 °C. (Wash soon after stopping run; if not possible, leave on device and wash next day.)
• Or follow the returns procedure to send the flow cell back to ONT. (Section 7, p. 29–30.)

Troubleshooting quick guide (selected items)

Documentation & records

Record the following in the run log (electronic or paper): sample ID(s); extraction method; DNA QC (Qubit, NanoDrop ratios), fragment size assessment; reagent kit lot/expiry; exact volumes and times used; bead ratios; library Qubit result and calculated loading mass/fmol; flow cell ID and Flow Cell Check pore count; priming/ loading time; MinKNOW run parameters (kit, basecalling setting, muxing, outputs); any deviations; troubleshooting actions; wash/return status.

Waste disposal

• Collect ethanol waste for appropriate disposal.
• Dispose of tips/tubes as laboratory waste per institutional policy.

References

• Oxford Nanopore Technologies, “Ligation sequencing DNA V14 (SQK‑LSK114) – MinION/GridION; GDE_9161_v114_revAB (26 Aug 2025)”. Page references in this SOP point to this document.

Appendix A — Quick‑reference reagent tables

A1. End‑prep (total 60 µL; p. 11) 47 µL DNA (+1 µL DCS optional) + 7 µL FFPE Buffer v2 + 2 µL FFPE Repair Mix + 3 µL Ultra II End‑Prep Enzyme → 20 °C 5 min → 65 °C 5 min → cool; AXP clean‑up as described.

A2. Ligation (total 100 µL; p. 15) 60 µL DNA + 5 µL LA + 25 µL LNB + 10 µL Salt‑T4 Ligase → 10 min RT; AXP 40 µL; wash ×2 with LFB or SFB; elute 15 µL EB (10 min; 37 °C optional).

A3. Flow‑cell priming mix with BSA (per FC; p. 19) FCF 1,170 µL + BSA 5 µL (50 mg/mL) + FCT 30 µL → 1,205 µL.

A4. Library loading mix (per FC; p. 23) SB 37.5 µL + LIB 25.5 µL (or LIS 25.5 µL) + DNA library 12 µL → 75 µL.

A5. Recommended flow‑cell loading amount (p. 16) <1 kb: 100 fmol; 1–10 kb: 35–50 fmol; >10 kb: 300 ng (or load entire library if yields are low).

Appendix B — Visual cues cited in this SOP

• p. 19 (table & diagram): Priming mix recipe; flow‑cell placement.
• p. 21 (diagram): Draw‑back 20–30 µL to remove bubbles.
• p. 22–25 (diagrams): Priming steps (800 µL + wait 5 min; second 200 µL prime), dropwise 75 µL loading into SpotON.
• p. 26–27 (diagrams): Light‑shield fitting and caution.