Beads-free ONT ligation kit library preparation for ultra-long read sequencing

Equipment

• Mini centrifuge
• Tabletop centrifuge
• Thermocycler 
• Heatblock
• Qubit
• MinION device and IT set up

Materials and consumables

• High quality HMW DNA e.g DNA extracted from Nanobind CBB Big DNA Kit (SKU NB-900-001-01, Circulomics) or equivalent
• Tris-HCl (pH-8)
• NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (E7180S, NEB)
• Ligation Sequencing Kit (SQK-LSK109, ONT)
• PEG solution (9%PEG 8k (w/v), 1 M NaCl, 10 mM Tris-HCl (pH-8) - [Polyethylene glycol: V3011, Promega; NaCl 71580-500G, Tris T6066-500G, Sigma]
• Short Read Elimiminator XL (SKU SS-100-111-01, Circulomics)
• Nuclease free water
• P200 wide bore pipette tips (LC1152-965, Adelab)
• Qubit dsDNA HS assay kit (Q32854, Life Technologies Australia) 
• MinION Flow Cell (FLO-MIN106, R9.4.1)
• Wide bore pipette tips (P200)
• 1.5 ml Low bind DNA Eppendrof tube
• Qubit tubes
• PCR tubes

A. DNA repair and end-prep (2 h and 15 min)

1. Thaw FFPE DNA Repair Buffer and End-prep reaction buffer and mix well by vertexing. Check if any white precipitate appeared.
2. Mix all reagents in a 0.2 ml thin-walled PCR tube and mix gently by using P200 wide-bore pipette tips. Try to avoid air bubbles formation which is hard to remove due to highly viscus solution and spin down in a mini centrifuge.

1. Using a thermocycler, incubate at 20 ºC for 10 min and 65 ºC for 10 min.
2. Transfer the reaction solution into a fresh DNA low bind 1.5 ml Eppendorf tube and add 60 µl of PEG/NaCl solution [9%PEG 8k (w/v), 1 M NaCl, 10 mM Tris-HCl (pH-8)] and mix well by tapping the tube followed by 30 min incubation at room temperature (RT).
3. Centrifuge the solution at 13k rpm at RT for 30 min. Note: Place the tube’s hinge facing away from the centre of centrifuge. While centrifuging the tube, prepare 1 ml 70% ethanol nuclease free water.
4. Remove the supernatant and add 200 µl of 70% ethanol without disturbing the DNA pellet and spin it at 13k for 5 min and repeat this step once.
5. Allow DNA to air dry for a couple of mins and then add 33 µl of EB buffer [10 mM Tris-HCl (pH 8.0)] and incubate at 37°C for 15 min or can leave overnight at 4 °C. Use 1 µl eluate for qubit quantification

B. Adaptor ligation and clean-up (1 h 45 min)

1. Spin down Adapter mix (AMX) and T4 Ligase from NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (E7180S).
2. Thaw Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vertexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
3. Thaw the Elution Buffer (EB) at room temperature, mix well by vertexing, spin down in mini centrifuge and place on ice.
4. Thaw L Fragment Buffer (LFB) at room temperature, mix well by vertexing, spin down in mini centrifuge and place on ice.
5. In a 1.5 ml DNA LowBind Eppendorf tube, mix the reagents in the following order.

1. Mix the reaction solution by pipetting (P200 wide bore or P100 wide bore or cut tip of the regular pipette tips) without introducing air bubbles and spin down. Incubate the reaction for 20 min at RT.
2. Add 60 µl of SRE XL buffer and mix gently by tapping the tube and centrifuge it at 13k for 30 min at RT. Note: Place the tube’s hinge facing away from the centre of centrifuge.
3. Pipette up the supernatant from other side of the tube’s hinge region without disturbing the DNA pellet. Add 250 µl Long LFB and spin it at 13k for 3 min at RT.
4. Repeat step 8 once.
5. Allow to dry 2-3 min, but do not completely dry the pellet that makes hard to dissolve DNA pellet.
6. Add 39 µl of EB buffer and incubate at 37 °C for 15 min or can leave overnight at 4 °C.
7. Use 1 µl eluate for qubit quantification.

C. Priming and loading library (1 h)

1. Follow the standard sequencing protocol for MinION except loading library preparation where loading beads (LB) was excluded to avoid clumping of ultra-HMW DNA on the beads.
2. Perform the nuclease flush while pore availability becomes 10-20% and load the fresh library to increase throughput.
3. Keep the washed flow cells if the pores are still available (>200 pores) after generating required data. 4-5 times nuclease flush can be done for a single flow cell.

Depending on the sample, 30-55% of DNA was recovered at final library which is enough for 2-4 times library top-up. From three different samples, average of ~ 400 kb longest reads was achieved through this method. 7 Gb was final yield in 24-36 h depending on the flow cells with one-two loading of nuclease flush and fresh library loading.

Table: Sequencing yield and stats of worked samples

*Used flow cell and sequencing was run for 48 h.

• OXFORD NANOPORE TECHNOLOGIES, Nanopore Protocol, Genomic DNA by ligation (SQK-LSK109) Version: GDE_9063_v109_revT_14Aug2019. 

• Jain, M., Koren, S., Miga, K. H., Quick, J., Rand, A. C., Sasani, T. A., Tyson, J. R., Beggs, A. D., Dilthey, A. T., Fiddes, I. T., Malla, S., Marriott, H., Nieto, T., O'grady, J., Olsen, H. E., Pedersen, B. S., Rhie, A., Richardson, H., Quinlan, A. R., Snutch, T. P., Tee, L., Paten, B., Phillippy, A. M., Simpson, J. T., Loman, N. J. & Loose, M. 2018. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol, 36, 338-345. https://www.nature.com/articles/nbt.4060

• Quick, J. (2018). Ultra-long read sequencing protocol for RAD004 V3. https://dx.doi.org/10.17504/protocols.io.mrxc57n

• Tyson, J. (2020). Rocky Mountain adventures in Genomic DNA sample preparation, ligation protocol optimisation / simplification and Ultra long read generation. dx.doi.org/10.17504/protocols.io.7euhjew