Ligation Sequencing DNA V14 (SQK-LSK114) Protocol

Protocol for Oxford Nanopore Technologies models including: SQK-LSK114, Ligation sequencing DNA, Oxford Nanopore Technologies, DNA sequencing, MinION, GridION, DNA repair, adapter ligation, library preparation, flow cell, sequencing protocol

Ligation sequencing DNA V14 (SQK-LSK114)

30 lug 2024 — INSTRUCTIONS. NOTES/OBSERVATIONS. DNA repair and end-prep. Check your flow cell. Thaw DNA Control Sample (DCS) at RT, spin down, mix by pipetting, and place on ...

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Ligation sequencing DNA V14 (SQK-LSK114)-minion

Ligation sequencing DNA V14 (SQK-LSK114)
Version: GDE_9161_v114_revW_29Jun2022 Last update: 21/08/2024
Flow Cell Number: ......................................................................................

DNA Samples: .........................................................................................

Before start checklist
Materials

Consumables

Equipment

1 µg (or 100-200 fmol) high molecular weight genomic DNA
OR 100+ ng high molecular weight genomic DNA if performing DNA fragmentation
Ligation Sequencing Kit V14 (SQK-LSK114)

MinION and GridION Flow Cell
Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7672S or E7672L)
Bovine Serum Albumin (BSA) (50 mg/ml) (e.g InvitrogenTM UltraPureTM BSA 50 mg/ml, AM2616)
Freshly prepared 80% ethanol in nucleasefree water
Nuclease-free water (e.g. ThermoFisher, AM9937)
1.5 ml Eppendorf DNA LoBind tubes
0.2 ml thin-walled PCR tubes
QubitTM Assay Tubes (Invitrogen, Q32856)

MinION or GridION device
MinION and GridION Flow Cell Light Shield
Hula mixer (gentle rotator mixer)
Magnetic rack, suitable for 1.5 ml Eppendorf tubes
Microfuge
Vortex mixer
Thermal cycler Ice bucket with ice Timer Qubit fluorometer (or equivalent for QC check) Pipettes and pipette tips P2, P10, P20, P100, P200, P1000

MINASSTSRFULCOTWIONS DNA repair and end-prep

NOTES/OBSERVATIONS

Check your flow cell. Thaw DNA Control Sample (DCS) at RT, spin down, mix by pipetting, and place on ice.

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DNA Samples: ......................................................................................... NOTES/OBSERVATIONS

Prepare the NEB reagents in accordance with manufacturer's instructions, and place on ice.
Thaw all reagents on ice.
Flick and/or invert the reagent tubes to ensure they are well mixed. Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
Vortex the FFPE DNA Repair Buffer v2, or the NEBNext FFPE DNA Repair Buffer and Ultra II End Prep Reaction Buffer to ensure they are well mixed. Note: These buffers may contain a white precipitate. If this occurs, allow the mixture(s) to come to RT and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.
The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

Prepare the DNA in Nuclease-free water: Transfer 1 g (or 100-200 fmol) input DNA into a 1.5 ml Eppendorf DNA LoBind tube. Adjust the volume to 47 l with Nuclease-free water. Mix thoroughly by pipetting up and down, or by flicking the tube. Spin down briefly in a microfuge.

In a 0.2 ml thin-walled PCR tube, mix the following: 47 µl DNA from the previous step 1 µl DNA CS (optional) 7 µl NEBNext FFPE DNA Repair Buffer v2 2 µl NEBNext FFPE DNA Repair Mix 3 µl Ultra II End-prep Enzyme Mix 47 µl DNA from the previous step 1 µl DNA CS (optional) 3.5 µl NEBNext FFPE DNA Repair Buffer 2 µl NEBNext FFPE DNA Repair Mix 3.5 µl Ultra II End-prep Reaction Buffer 3 µl Ultra II End-prep Enzyme Mix

Thoroughly mix the reaction by gently pipetting and briefly spinning down.

Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.

Resuspend the AMPure XP Beads (AXP) by vortexing.

Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.

Add 60 µl of resuspended the AMPure XP Beads (AXP) to the end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.

Prepare 500 l of fresh 80% ethanol in Nuclease-free water.

Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant.

Keep the tube on the magnet and wash the beads with 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

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DNA Samples: ......................................................................................... NOTES/OBSERVATIONS

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 61 µl Nuclease-free water. Incubate for 2 minutes at RT.

Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.

Remove and retain 61 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of eluted sample using a Qubit fluorometer.

Take forward the repaired and end-prepped DNA into the adapter ligation step. However, at this point it is also possible to store the sample at 4°C overnight.

Adapter ligation and clean-up

IMPORTANT
Although third-party ligase products may be supplied with their own buffer, the ligation efficiency of the Ligation Adapter (LA) is higher when using the Ligation Buffer (LNB) supplied in the Ligation Sequencing Kit.
Spin down the Ligation Adapter (LA) and Salt-T4® DNA Ligase, and place on ice.
Thaw Ligation Buffer (LNB) at RT, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
Thaw the Elution Buffer (EB) at RT and mix by vortexing. Then spin down and place on ice.
IMPORTANT
Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally.
To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB) To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB)
Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at RT and mix by vortexing. Then spin down and place on ice.
In a 1.5 ml Eppendorf DNA LoBind tube, mix in the following order: 60 µl DNA sample from the previous step 5 µl Ligation Adapter (LA) 25 µl Ligation Buffer (LNB) 10 µl Salt-T4® DNA Ligase
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 10 minutes at RT.
Resuspend the AMPure XP Beads (AXP) by vortexing.

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DNA Samples: ......................................................................................... NOTES/OBSERVATIONS

Add 40 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.

Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant when clear and colourless.

Wash the beads by adding either 250 l Long Fragment Buffer (LFB) or 250 l Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat the previous step.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at RT. For high molecular weight DNA, incubating at 37°C can improve the recovery of long fragments.

Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.

Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

Quantify 1 µl of eluted sample using a Qubit fluorometer.

Depending on your DNA library fragment size, prepare your final library in 12 µl of Elution Buffer (EB). 100 fmol Very short (<1 kb) 3550 fmol Short (1-10 kb) 300 ng Long (>10 kb)
The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.
Priming and loading the MinION and GridION Flow Cell

IMPORTANT
Please note, this kit is only compatible with R10.4.1 flow cells (FLO-MIN114).

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at RT before mixing by vortexing. Then spin down and store on ice.
IMPORTANT
For optimal sequencing performance and improved output on MinION R10.4.1 flow cells (FLO-MIN114), we recommend adding Bovine Serum Albumin (BSA) to the flow cell priming mix at a final concentration of 0.2 mg/ml.
To prepare the flow cell priming mix with BSA, combine Flow Cell Flush (FCF) and Flow Cell Tether (FCT), as directed below. Mix by pipetting at RT.
1,170 µl Flow Cell Flush (FCF) 5 µl Bovine Serum Albumin (BSA) at 50 mg/ml 30 µl Flow Cell Tether (FCT)

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DNA Samples: .........................................................................................

MINASSTSRFULCOTWIONS

NOTES/OBSERVATIONS

Open the MinION or GridION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Slide the flow cell priming port cover clockwise to open the priming port.
IMPORTANT
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
Set a P1000 pipette to 200 µl Insert the tip into the priming port Turn the wheel until the dial shows 220-230 µl, to draw back 20-30 µl, or until you can see a small volume of buffer entering the pipette tip
Note: Visually check that there is continuous buffer from the priming port across the sensor array.
Load 800 µl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for five minutes. During this time, prepare the library for loading by following the steps below.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
IMPORTANT
The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows: 37.5 µl Sequencing Buffer (SB) 25.5 µl Library Beads (LIB) mixed immediately before use, or Library Solution (LIS), if using 12 µl DNA library
Complete the flow cell priming: Gently lift the SpotON sample port cover to make the SpotON sample port accessible. Load 200 µl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
Mix the prepared library gently by pipetting up and down just prior to loading.
Add 75 l of the prepared library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.
IMPORTANT
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.

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DNA Samples: .........................................................................................

MINASSTSRFULCOTWIONS

NOTES/OBSERVATIONS

Place the light shield onto the flow cell, as follows:
Carefully place the leading edge of the light shield against the clip. Note: Do not force the light shield underneath the clip.
Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.

Close the device lid and set up a sequencing run on MinKNOW.
Flow cell reuse and returns
After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.

Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.
IMPORTANT
If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.

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Prawn Prawn

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Ligation sequencing DNA V14 (SQK-LSK114)

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