Ligation sequencing gDNA V14 — human sample (N50 10 kb) on PromethION (SQK-LSK114)

Oxford Nanopore Technologies
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PromethION: Protocol

Ligation sequencing gDNA V14 — human sample (N50 10 kb) on PromethION (SQK-LSK114) V GDH_9173_v114_revP_10Nov2022

  • This protocol uses N50 of 10 kb genomic DNA extracted from human cell lines
  • Sample preparation time: ~220 minutes and library preparation time: ~60 minutes
  • Data analysis: 1-2 hours
  • No PCR
  • Compatible with R10.4.1 flow cells

For Research Use Only

FOR RESEARCH USE ONLY

Overview

  • This protocol uses N50 of 10 kb genomic DNA extracted from human cell lines
  • Sample preparation time: ~220 minutes and library preparation time: ~60 minutes
  • Data analysis: 1-2 hours
  • No PCR
  • Compatible with R10.4.1 flow cells

For Research Use Only

Document version: GDH_9173_v114_revP_10Nov2022

1. Overview of the protocol

Introduction to the protocol for sequencing 10 kb human DNA on PromethION using the Ligation Sequencing Kit V14 (SQK-LSK114)

This protocol describes an end-to-end process to prepare, sequence gDNA from cultured cells samples and analyse using the “Human variant workflow” in Epi2Me. The identification of structural variants (SVs) and single nucleotide variants (SNVs) play a pivotal role in our understanding of genetic diversity, disease mechanisms, and evolutionary biology1,2. The protocol aims to produce libraries with a read N50 ~10 kb and generate ~30-40x coverage of the genome, to provide robust calling of small and large variants as well as provide methylation and phasing information.

Briefly, genomic DNA is extracted from 5 million cultured cells, and is extracted using the QIAGEN Puregene Cell Kit. DNA is then sheared with the Covaris g-TUBE™, and libraries prepared with the Ligation Sequencing Kit V14 (SQK-LSK114). Libraries are then sequenced on PromethION.

Data is basecalled and aligned by MinKNOW and the aligned BAM output data is analysed using the wf-human-variation workflow which uses Sniffles2, Clair3 and modbam2bed software to call structural variants (SVs), single nucleotide polymorphisms (SNPs) and for reporting DNA methylation.

Steps in the sequencing workflow:

Prepare for your experiment

You will need to:

  • Extract your input sample (cells)
  • Ensure you have your sequencing kit, the correct equipment, and third-party reagents
  • Download the software for acquiring and analysing your data
  • Check your flow cell to ensure it has enough pores for a good sequencing run

Sample preparation

Using the outlined extraction method, extract the gDNA from your cells, and fragment your gDNA using the Covaris g-TUBE.

Check the length, quantity and purity of your extracted material. The quality checks performed during the protocol are essential in ensuring experimental success.


Library preparation

The table below is an overview of the steps required in the library preparation, including timings and optional stopping points.

Library preparation Process Time Stop option
DNA repair and end-prep Repair and prepare the DNA ends for adapter attachment 35 minutes 4°C overnight
Adapter ligation and clean-up Attach the sequencing adapters to the DNA ends 20 minutes 4°C short-term storage or for repeated use, such as re-loading your flow cell
-80°C for single-use, long-term storage.
We strongly recommend sequencing your library as soon as it is adapted.
Priming and loading the flow cell Prime the flow cell and load the prepared library for sequencing 5 minutes

10 kb lsk114 workflow prom v2

Sequencing and analysis

You will need to:

  • Start a sequencing run using the MinKNOW software which will collect raw data from the device and convert it into basecalled reads.
  • Analyse data using the wf-human-variation workflow.
IMPORTANT

Compatibility of this protocol

This protocol should only be used in combination with:

2. Equipment and consumables

Materials
  • (FOR EXTRACTION) 5 x 10⁶ cells (e.g. cell culture or tissue sample)
  • (FOR LIBRARY PREPARATION) 1 µg of fragmented gDNA
  • Ligation Sequencing Kit V14 (SQK-LSK114)
Consumables
  • PromethION Flow Cell
  • NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7672S or E7672L)
  • Puregene Cell Kit (QIAGEN, 158043)
  • g-TUBE™ (Covaris, 520079)
  • 15 ml Falcon tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • 0.2 ml thin-walled PCR tubes
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Isopropanol, 100% (Fisher, 10723124)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
  • (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
  • Inoculation loop or disposable tweezers
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
Equipment
  • PromethION sequencing device
  • PromethION Flow Cell Light Shield
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack, suitable for 1.5 ml Eppendorf tubes
  • Incubator or water bath set at 37°C and 50°C
  • Centrifuge and rotor suitable for 15 ml Falcon tubes
  • Microfuge
  • Vortex mixer
  • Thermal cycler
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • P2 pipette and tips
  • Ice bucket with ice
  • Timer
  • Agilent Femto Pulse System (or equivalent for read length QC)
  • Qubit fluorometer (or equivalent for QC check)
IMPORTANT

The above list of materials, consumables, and equipment is for the extraction method in the sample preparation section, as well as the library preparation section of the protocol. If you have pre-extracted sample(s), you will only require the materials for the library preparation section of this protocol.

For the library preparation protocol, you will need 3 µg of genomic DNA with an N50 of 10 kb.

For this end-to-end workflow, we recommend extracting high molecular weight human gDNA from from 5 x 10⁶ cells (e.g. cell culture or tissue sample) using the QIAGEN Puregene Cell Kit in the Sample Preparation step.

Other extraction protocols are available but have not been tested by Oxford Nanopore Technologies.

Input DNA

How to QC your input DNA

It is important that the input DNA meets the quantity and quality requirements. Using too little or too much DNA, or DNA of poor quality (e.g. highly fragmented or containing RNA or chemical contaminants) can affect your library preparation.

For instructions on how to perform quality control of your DNA sample, please read the Input DNA/RNA QC protocol.

Chemical contaminants

Depending on how the DNA is extracted from the raw sample, certain chemical contaminants may remain in the purified DNA, which can affect library preparation efficiency and sequencing quality. Read more about contaminants on the Contaminants page of the Community.

TIP

Eppendorf tube orientation in centrifuge

For all centrifugation steps, ensure that tubes are loaded into the centrifuge with the hinge side of the tube facing outwards. This will assist in visual identification of the pellet.

Ensure gentle handling when removing the tubes from the centrifuge to avoid dislodging the pellet.

Eppendorf tube hinge into centrifuge image

TIP

We recommend using the NEBNext® Companion Module v2 for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7672S or E7672L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.

The previous version, NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB, E7180S or E7180L) is also compatible, but the recommended v2 module offers more efficient dA-tailing and ligation.

Third-party reagents

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore Technologies.

For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

Check your flow cell

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment. This should be done within 12 weeks of purchasing for MinION/GridION/PromethION or within four weeks of purchasing Flongle Flow Cells. Oxford Nanopore Technologies will replace any flow cell with fewer than the number of pores in the table below, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed. To do the flow cell check, please follow the instructions in the Flow Cell Check document.

Flow cell Minimum number of active pores covered by warranty
Flongle Flow Cell 50
MinION/GridION Flow Cell 800
PromethION Flow Cell 5000
IMPORTANT

We strongly recommend using the Ligation Buffer (LNB) supplied in the Ligation Sequencing Kit V14 rather than any third-party ligase buffers to ensure high ligation efficiency of the Ligation Adapter (LA).

IMPORTANT

Ligation Adapter (LA) included in this kit and protocol is not interchangeable with other sequencing adapters.

Ligation Sequencing Kit V14 (SQK-LSK114) contents

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format.

Single-use tubes format: SQK-LSK114 v2

Bottle format: SQK-LSK114 v3

Note: This Product Contains AMPure XP Reagent Manufactured by Beckman Coulter, Inc. and can be stored at -20°C with the kit without detriment to reagent stability.

Note: The DNA Control Sample (DCS) is a 3.6 kb standard amplicon mapping the 3' end of the Lambda genome.

3. Human cell line DNA extraction using the QIAGEN Puregene Cell Kit

Materials
  • 5 x 10⁶ cells (e.g. cell culture or tissue sample)
Consumables
  • Puregene Cell Kit (QIAGEN, 158043)
  • Freshly prepared cold 70% ethanol in nuclease-free water
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) (Fisher scientific, 10224683)
  • Phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 10010023)
  • Isopropanol, 100% (Fisher, 10723124)
  • 15 ml Falcon tubes
  • 2 ml Eppendorf DNA LoBind tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Inoculation loop or disposable tweezers
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
Equipment
  • Incubator or water bath set at 37°C and 50°C
  • Centrifuge and rotor suitable for 15 ml Falcon tubes
  • Vortex mixer
  • Wide-bore pipette tips
  • P1000 pipette and tips
  • P200 pipette and tips
  • Ice bucket with ice
  • Qubit fluorometer (or equivalent for QC check)

Prepare a 1.5 ml Eppendorf DNA LoBind tube with approximately 1 ml of 70% ethanol and store on ice to cool.

We recommend to harvest and pellet 5 x 10⁶ cells in a 1.5 ml Eppendorf DNA LoBind tube. However, this may vary depending on your sample type. If any liquid remains associated with the pellet, spin down the cells again, then aspirate and discard the remaining supernatant.

Add 200 µl of 1x PBS to pelleted cells.

Centrifuge at 300 x g for 3 minutes.

Without disturbing the pellet, aspirate and discard the supernatant.

Add 3 ml of Cell Lysis Solution, and pipet up and down gently to lyse the cells and homogenise the solution until no clumps remain.

Ensure that the solution is homogenous.

Transfer the resuspended cells to a 15 ml Falcon. If clumps of cells remain, gently invert the tube to ensure resuspension.

Incubate the sample at 37°C for 30 minutes.

Add 1000 µl of the Protein Precipitation Solution to the lysed cells and mix by vortexing for three pulses of 5 seconds.

Centrifuge the sample at 2000 x g for 5 minutes.

The precipitated protein should form a tight, reddish-brown pellet.

If the protein pellet is not tight, incubate on ice for 5 minutes and repeat the centrifugation.

Add 3 ml of isopropanol into a clean 15 ml falcon tube.

Mix by gently inverting the tube 50 times until the DNA is visible as threads or a clump.

Spool the DNA using an inoculation loop or disposable tweezers.

Briefly dip the spooled DNA in the 1.5 ml Eppendorf DNA LoBind tube containing cold 80% ethanol and allow to air dry for a few seconds.

Transfer the inoculation loop or tweezers with the spooled DNA to a 1.5 ml Eppendorf DNA LoBind tube containing 250 µl TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and allow the DNA to gently dislodge from the loop/tweezers.

Incubate the DNA pellet for 2 hours at 50°C, occasionally mixing the tube by gentle inversion to aid dissolving the pellet. Alternatively, the DNA pellet can be left overnight at room temperature.

Note: The pellet may take some time to dissolve. Ensure the solution is homogenous before proceeding to quantification by gentle inversion and spin down.

Quantify the sample three times using the Qubit dsDNA BR Assay Kit, ensuring that replicate Qubit measurements are consistent before continuing to the next step.

If the Qubit measurements are not consistent, this could indicate that the DNA has not been homogeneously resuspended. If this occurs, we recommend increasing the incubation time to aid with resuspension of the DNA pellet.

END OF STEP

Take the sample in 250 µl TE buffer forwards into the next step or the sample can be stored at 4°C overnight.

4. Shearing DNA for 10 kb input using the Covaris g-TUBE™

Materials
  • 2 µg of extracted high molecular weight gDNA
Consumables
  • g-TUBE™ (Covaris, 520079)
  • Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • (Optional) TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)
  • Qubit dsDNA BR Assay Kit (Invitrogen, Q32850)
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Agilent Genomic DNA 165 kb Analysis Kit (Agilent, FP-1002-0275)
Equipment
  • Microfuge
  • P200 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P2 pipette and tips
  • Agilent Femto Pulse System (or equivalent for read length QC)
  • Qubit fluorometer (or equivalent for QC check)
OPTIONAL ACTION

We recommend using TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) rather than nuclease-free water if the library is going to be stored over a long-term period.

Transfer 2 μg extracted gDNA into a 1.5 ml Eppendorf DNA LoBind tube, and adjust the volume to 100 μl with nuclease-free water.

Mix the DNA thoroughly by flicking the tube. Spin down briefly in a microfuge.

Transfer the genomic DNA sample in 100 μl to a Covaris g-TUBE™.

For a fragment length of 10 kb, centrifuge the g-TUBE™ at 4300 x g for one minute at room temperature. Remove and check that all the DNA has passed through the tube.

Note: Please ensure that you are using the correct speed for your equipment and input sample to achieve shearing of 10 kb fragment lengths.

TIP

If DNA remains in the upper chamber of the Covaris g-TUBE™, spin again for one minute at the same speed.

Invert the g-TUBE™ and centrifuge again for one minute to collect the fragmented DNA. Remove and check that all the DNA has passed through the tube.

TIP

If DNA remains in the upper chamber of the Covaris g-TUBE™, spin again for one minute at the same speed.

Transfer the 100 μl fragmented DNA into a clean 1.5 ml Eppendorf DNA LoBind tube.

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer and check sample read length using the Agilent Femto Pulse System. An N50 of 10 kb read lengths is to be expected.

END OF STEP

Take forwards 1 μg sample into the next step or the sample can be stored at 4°C overnight.

5. DNA repair and end-prep

Materials
  • 1 µg of fragmented gDNA
  • AMPure XP Beads (AXP)
Consumables
  • NEBNext® FFPE DNA Repair Mix (M6630) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
  • NEBNext® FFPE DNA Repair Buffer v2 (E7363) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
  • NEBNext® Ultra II End Prep Enzyme Mix (E7646) from the NEBNext® Companion Module v2 (NEB, E7672S or E7672L)
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
  • Nuclease-free water (e.g. ThermoFisher, AM9937)
  • Freshly prepared 80% ethanol in nuclease-free water
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • 0.2 ml thin-walled PCR tubes
  • 1.5 ml Eppendorf DNA LoBind tubes
Equipment
  • P1000 pipette and tips
  • P100 pipette and tips
  • P10 pipette and tips
  • Microfuge
  • Thermal cycler
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Ice bucket with ice
Optional equipment
  • Qubit fluorometer (or equivalent for QC check)
CHECKPOINT

Check your flow cell.

We recommend performing a flow cell check before starting your library prep to ensure you have a flow cell with enough pores for a good sequencing run.

See the flow cell check instructions in the MinKNOW protocol for more information.

Prepare the NEB reagents in accordance with manufacturer’s instructions, and place on ice.

For optimal performance, NEB recommend the following:

  1. Thaw all reagents on ice.

  2. 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.

  3. Always spin down tubes before opening for the first time each day.

  4. 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 room temperature and pipette the buffer several times to break up the precipitate, followed by a quick vortex to mix.

  5. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.

Prepare the DNA in nuclease-free water:

  1. Transfer 1 µg of 10 kb fragmented gDNA from the sample extraction into a 0.2 ml thin-walled PCR tube.

  2. Adjust the volume to 48 μl with nuclease-free water.

  3. Mix thoroughly by pipetting up and down, or by flicking the tube.

  4. Spin down briefly in a microfuge.

In the 0.2 ml thin-walled PCR tube containing your gDNA, mix the following:

Reagent Volume
DNA from the previous step 48 µl
NEBNext FFPE DNA Repair Buffer v2 7 µl
NEBNext FFPE DNA Repair Mix 2 µl
Ultra II End-prep Enzyme Mix 3 µl
Total 60 µl

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

Using a thermal cycler, incubate the reaction at 20°C for 5 minutes, then 65°C for 5 minutes and hold at 4°C.

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 room temperature.

Prepare 600 µl of fresh 80% ethanol in nuclease-free water.

Spin down the sample and pellet on a magnet for 10 minutes 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 250 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

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 room temperature.

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.

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

END OF STEP

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.

6. Adapter ligation and clean-up

Materials
  • Ligation Adapter (LA)
  • Ligation Buffer (LNB)
  • Long Fragment Buffer (LFB)
  • AMPure XP Beads (AXP)
  • Elution Buffer (EB) from the Ligation Sequencing Kit
Consumables
  • Salt-T4® DNA Ligase (NEB, M0467)
  • 1.5 ml Eppendorf DNA LoBind tubes
  • Qubit™ Assay Tubes (Invitrogen, Q32856)
  • Qubit dsDNA HS Assay Kit (Invitrogen, Q32851)
Equipment
  • Hula mixer (gentle rotator mixer)
  • Magnetic rack
  • Microfuge
  • Vortex mixer
  • P1000 pipette and tips
  • P100 pipette and tips
  • P20 pipette and tips
  • P10 pipette and tips
  • Qubit fluorometer (or equivalent for QC check)
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 room temperature, 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 room temperature and mix by vortexing. Then spin down and place on ice.

Thaw the Long Fragment Buffer (LFB) at room temperature 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:

Between each addition, pipette mix 10-20 times.

Reagent Volume
DNA sample from the previous step 60 µl
Ligation Adapter (LA) 5 µl
Ligation Buffer (LNB) 25 µl
Salt-T4® DNA Ligase 10 µl
Total 100 µl

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

Incubate the reaction for 10 minutes at room temperature.

Resuspend the AMPure XP Beads (AXP) by vortexing.

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 room temperature.

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 250 μl Long Fragment Buffer (LFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet for at least 5 minutes. Remove the supernatant using a pipette and discard.

Note: Take care when removing the supernatant, the viscosity of the buffer can contribute to loss of beads from the pellet.

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 33 µl Elution Buffer (EB). Spin down and incubate for 10 minutes at 37°C.

Pellet the beads on a magnet for 10 minutes until the eluate is clear and colourless.

Remove and retain 33 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

Dispose of the pelleted beads

CHECKPOINT

Quantify 1 µl of eluted sample using a Qubit fluorometer.

IMPORTANT

For libraries with an N50 of 10 kb, we recommend loading at least 200-300 ng (35-50 fmol) of your final prepared library onto the R10.4.1 flow cell.

This protocol has been optimised for output to achieve the analysis of one genome at 30X coverage.

It is vital to ensure 200-300 ng of 10 kb input is loaded onto the flow cell to maximise pore occupancy from the beginning of the run for optimal sequencing. If pore occupancy is low at the beginning of a run, more terminal blocking will occur throughout sequencing which will result in lower outputs.

If required, we recommend using a mass to mol calculator such as the NEB calculator.

END OF STEP

The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.

TIP

Library storage recommendations

We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short-term storage or repeated use, for example, re-loading flow cells between washes. For single use and long-term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes.

7. Priming and loading the PromethION Flow Cell

Materials
  • Sequencing Buffer (SB)
  • Library Beads (LIB)
  • Library Solution (LIS)
  • Flow Cell Tether (FCT)
  • Flow Cell Flush (FCF)
Consumables
  • PromethION Flow Cell
  • 1.5 ml Eppendorf DNA LoBind tubes
Equipment
  • PromethION device
  • PromethION Flow Cell Light Shield
  • P1000 pipette and tips
  • P200 pipette and tips
  • P20 pipette and tips
IMPORTANT

This kit is only compatible with R10.4.1 flow cells (FLO-PRO114M).

Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at room temperature before mixing by vortexing. Then spin down and store on ice.

To prepare the flow cell priming mix, combine Flow Cell Tether (FCT) and Flow Cell Flush (FCF), as directed below. Mix by vortexing at room temperature.

Note: We are in the process of reformatting our kits with single-use tubes into a bottle format. Please follow the instructions for your kit format.

Single-use tubes format: Add 30 µl Flow Cell Tether (FCT) directly to a tube of Flow Cell Flush (FCF).

Bottle format: In a clean suitable tube for the number of flow cells, combine the following reagents:

Reagent Volume per flow cell
Flow Cell Flush (FCF) 1,170 µl
Flow Cell Tether (FCT) 30 µl
Total volume 1,200 µl
IMPORTANT

After taking flow cells out of the fridge, wait 20 minutes before inserting the flow cell into the PromethION for the flow cell to come to room temperature. Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.

For PromethION 2 Solo, load the flow cell(s) as follows:

  1. Place the flow cell flat on the metal plate.

  2. Slide the flow cell into the docking port until the gold pins or green board cannot be seen.

J2068 FC-into-P2-animation V5

For the PromethION 24/48, load the flow cell(s) into the docking ports:

  1. Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position.
  2. Press down firmly onto the flow cell and ensure the latch engages and clicks into place.

Step 1a V3

Step 1B

IMPORTANT

Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.

Screenshot 2021-04-08 at 12.08.37

Slide the inlet port cover clockwise to open.

Prom loading 2

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 inlet port, draw back a small volume to remove any air bubbles:

  1. Set a P1000 pipette tip to 200 µl.
  2. Insert the tip into the inlet port.
  3. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.

Step 3 v1

Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes. During this time, prepare the library for loading using the next steps in the protocol.

Step 4 v1

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.

We recommend using the Library Beads (LIB) for most sequencing experiments. However, the Library Solution (LIS) is available for more viscous libraries.

In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows:

Reagent Volume per flow cell
Sequencing Buffer (SB) 100 µl
Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS) 68 µl
DNA library 32 µl
Total 200 µl

Note: Library loading volume has been increased to improve array coverage.

Complete the flow cell priming by slowly loading 500 µl of the priming mix into the inlet port.

Step 5 v1

Mix the prepared library gently by pipetting up and down just prior to loading.

Load 200 µl of library into the inlet port using a P1000 pipette.

Step 6 v1

Close the valve to seal the inlet port.

Step 7 V2

IMPORTANT

Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.

We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps. The shield can be removed when the library has been removed from the flow cell.

If the light shield has been removed from the flow cell, install the light shield as follows:

  1. Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID.
  2. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.

J2264 - Light shield animation PromethION Flow Cell 8a FAW

J2264 - Light shield animation PromethION Flow Cell 8b FAW

END OF STEP

Close the PromethION lid when ready to start a sequencing run on MinKNOW.

Wait a minimum of 10 minutes after loading the flow cells onto the PromethION before initiating any experiments. This will help to increase the sequencing output.

8. Data acquisition and basecalling

How to start sequencing

The sequencing device control, data acquisition and real-time basecalling are carried out by the MinKNOW software.

We recommend basecalling with the high accuracy (HAC) basecaller in real-time with BAM selected as output type using the P2 Solo or P24/P48 device.

You must generate a BAM file from your sequening run, as this is required for input into the wf-human-variation workflow.

Refer to the links below containing the detailed instructions for setting up the device and sequencing run:

Below are the recommended sequencing parameters for MinKNOW.

MinKNOW settings for 10kb human sample on PromethION

We recommend using the modified bases option for basecalling and ensuring a BAM output is selected when setting up your MinKNOW run. The remaining sequencing parameters are kept to default. Below are our current recommendations:

Positions

Flow cell position: [user defined]

Experiment name: [user defined]

Flow cell type: FLO-PRO114M

Sample ID: [user defined]

Kit

Kit selection: Ligation Sequencing Kit (SQK-LSK114)

Run configuration

Sequencing and analysis

Basecalling: On [default] Modified bases: On with '5mC & 5hmC CG contexts' selected Model: High-accuracy basecalling (HAC) [default]

Barcoding: Disabled [default]

Alignment: Off [default]
We do not currently recommend live alignment during sequencing, as it can slow down system processing.

Adaptive sampling: Off [default]

Advanced options Active channel selection: On [default] Time between pore scans: 1.5 [default] Reserve pores: On [default]

Data targets

Run limit: 72 hours [default]

Output

Output format .POD5: On [default] .FASTQ: On [default] .BAM: On

Filtering: On [default] Qscore: 9 [default] Minimum read length: 200 bp [default]

IMPORTANT

We do not recommend live alignment during sequencing, as it can slow down system processing.

You can align your BAM file post-sequencing by following one of the methods below:

Aligning the BAM file in MinKNOW Aligning the BAM file during the wf-human-variation workflow
Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing.

The aligned BAM file can be used as your file input in the wf-human-variation workflow.

Using mapped BAM as input, the workflow will take 1-2 hours.		 You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up.

Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours.

Further information is available in the Downstream analysis section of this protocol.

9. Downstream analysis

Analysis of human cell DNA sequence data

For the analysis of human cell DNA sequence data, we recommend the wf-human-variation workflow. This end-to-end software pipeline is implemented using the Nextflow workflow language and implements methods for the calling of single nucleotide polymorphisms (SNPs), structural variants (SVs), and for reporting DNA methylation information.

The wf-human-variation workflow is best run from the BAM file produced by MinKNOW when the modified base model for basecalling is selected. If sequence read mapping to the reference genome is not performed by MinKNOW, we recommend to perform the basecalling using the wf-basecalling workflow. Ensure you save the outputs in BAM format by providing the --output_bam option.

The tools below are used in the analysis workflow and can be run in isolation or together:

  1. Sniffles2 calls SVs and file output include an HTML report of QC metrics and VCF format list of variants and their quality scores.

  2. Clair3 calls SNPs and file output includes an HTML report of QC metrics and VCF format list of variants and their quality scores.

  3. modkit extracts methylation information from the provided BAM file which is summarised in a BEDmethyl format file.

The wf-human-variation workflow is preconfigured using appropriate parameters and requires tuning only for the choice of reference genome and Clair3 model. Please see the project’s documentation for further details.

The results from the wf-human-variation workflow can be further explored by viewing in a track-based genome browser such as IGV can be assessed for known pathogenicity through tertiary analysis software.

EPI2ME analysis workflow

The wf-human-variation workflow is intended to be run using the Nextflow software.

We recommend performing downstream analysis using EPI2ME which facilitates bioinformatic analyses by allowing users to run Nextflow workflows in a Graphical User Interfaces (GUI). EPI2ME maintains a collection of bioinformatic workflows which are curated and actively maintained by experts in long-read sequence analysis. The collection of all our available EPI2ME workflows can be found here.

For new users, the quick start guide can be found here outlining how to use this interface.

IMPORTANT

Compute requirements for the wf-human-variation workflow on EPI2ME

Recommended requirements Minimum requirements
CPUs = 32 CPUs = 16
Memory = 128GB Memory = 32GB

Approximate run time: Variable depending on whether it is targeted sequencing or whole genome sequencing, as well as coverage and the individual analyses requested. For instance, a 90X human sample run (options: --snp --sv --mod --str --cnv --phased --sex male) takes less than 8h with recommended resources.

ARM processor support: False

OPTIONAL ACTION

The wf-human-variation workflow can also be run using the command line interface (CLI)

Please see the Github page for further details.

Note: We only recommend the command line interface (CLI) for experienced users.

Open the EPI2ME app using the desktop shortcut.

On the landing page, open the workflow tab on the left-hand sidebar.

1 landing page EPI2ME

Navigate to the Available workflows tab and click on wf-human-variation option.

2 wf human var

Click install.

3 install human var

Navigate to the Installed tab and click on the installed wf-human-variation workflow.

4 wf human var select

OPTIONAL ACTION

If the workflow was already installed, check for updates by clicking 'Update workflow'.

We recommend running the latest version of our workflows for the best results.

5 wf human var update

Click on Run this workflow to open the launch wizard.

6 human var run

Select the environment you are running the workflow from:

7 human var run II

Click on the sub-workflow(s) you want to run in the wf-human-variation analysis.

8 wf human var subworkflows

You must have at least one of the sub-workflows selected to proceed with analysis.

9 wf human var subworkflows selected

Note: For more information on the sub-workflows click on the "Expand" option in the platform, or visit our online EPI2ME documentation.

Navigate to the 'Main options' tab to assign a 'Sample name' as an identifier in workflow outputs.

10 wf human var sample name

IMPORTANT

The wf-human-variation workflow uses sequencing data in the form of a single BAM file or a folder of BAM files.

The BAM files used as an input can be aligned or unaligned:

Aligning the BAM file in MinKNOW (prior to the wf-human-variation workflow) Aligning the BAM file during the wf-human-variation workflow (during the wf-human-variation workflow)
Align the BAM output after live basecalling in MinKNOW. This will prevent slowing down your sytems processing.

The aligned BAM file can be used as your file input in the wf-human-variation workflow.

For more information on post-run alignment in MinKNOW please visit our MinKNOW protocol.

Using mapped BAM as input, the workflow will take 1-2 hours.		 You can provide a reference genome along with the unaligned BAM file during the wf-human-variation workflow set-up.

Using an unmapped BAM is used as input, the workflow will take approximately 5-8 hours.

In the 'Main options' upload your sequencing data in the form of a single BAM file or a folder of BAM files.

11 wf human var BAM input

OPTIONAL ACTION

If you have an unaligned BAM file as input, in the 'Main options' upload your reference genome in FASTA format.

12 wf human var reference FASTA

Click Launch workflow.

Ensure all parameter options have green ticks.

13 wf human var launch

Once the wf-human-variation workflow finishes, a report will be produced alongside output files.

14 wf human var outputs

wf-human-variation workflow outputs

The primary workflow outputs include:

  • gzipped VCF file containing the SNPs in the dataset from --snp
  • gzipped VCF file containing the SVs in the dataset from --sv
  • gzipped bedMethyl file aggregating modified base counts from --mod
  • HTML report detailing the primary findings of the workflow for QC metrics, and SNP and SV calling
  • If an unaligned BAM file was provided, the workflow will ouput a CRAM file containing the alignments used to make the downstream variant calls.

The secondary workflow outputs:

  • mosdepth ouputs include:
    • {sample_name}.mosdepth.global.dist.txt: a cumulative distribution indicating the proportion of total bases for each and all reference sequences
    • {sample_name}.regions.bed.gz: the mean coverage for each region in the provided BED file
    • {sample_name}.thresholds.bed.gz: the number of bases in each region that are covered at or above each threshold value (1, 10, 20, 30X)

  • bamstats ouputs include:
    • {sample_name}.readstats.tsv.gz: a gzipped TSV summarising per-alignment statistics produced by bamstats
    • {sample_name}.ftagstat.tsv: a text file with summary alignment statistics for each reference sequence

wf-human-variation workflow tips

It is possible to phase SNPs, SVs and modified bases by providing the --phased option.

To improve the accuracy of SV calling, specify a suitable tandem repeat BED for your reference with --tr_bed.

Aggregation of methylation calls with --mod requires data to be basecalled with a model that includes base modifications, providing the MM and ML BAM tags. To do so on MinKNOW, ensure 'Modified bases' option is selected during basecalling set up, with the '5mC' model selected.

Ensure to retain the input reference when basecalling or alignment is performed as CRAM files cannot be read without the corresponding input reference.

For a full list of available basecalling models, refer to the Dorado documentation.

10. Flow cell reuse and returns

Materials
  • Flow Cell Wash Kit (EXP-WSH004)

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°C to +8°C.

The Flow Cell Wash Kit protocol is available on the Nanopore Community.

TIP

We recommend you to wash the flow cell as soon as possible after you stop the run. However, if this is not possible, leave the flow cell on the device and wash it the next day.

Alternatively, follow the returns procedure to send the flow cell back to Oxford Nanopore.

Instructions for returning flow cells can be found here.

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.

11. Issues during DNA extraction and library preparation

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Low sample quality

Observation Possible cause Comments and actions
Low DNA purity (Nanodrop reading for DNA OD 260/280 is <1.8 and OD 260/230 is <2.0–2.2) The DNA extraction method does not provide the required purity The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Consider performing an additional AMPure bead clean-up step.

Low DNA recovery after AMPure bead clean-up

Observation Possible cause Comments and actions
Low recovery DNA loss due to a lower than intended AMPure beads-to-sample ratio 1. AMPure beads settle quickly, so ensure they are well resuspended before adding them to the sample.

2. When the AMPure beads-to-sample ratio is lower than 0.4:1, DNA fragments of any size will be lost during the clean-up.
Low recovery DNA fragments are shorter than expected The lower the AMPure beads-to-sample ratio, the more stringent the selection against short fragments. Please always determine the input DNA length on an agarose gel (or other gel electrophoresis methods) and then calculate the appropriate amount of AMPure beads to use. SPRI cleanup
Low recovery after end-prep The wash step used ethanol <70% DNA will be eluted from the beads when using ethanol <70%. Make sure to use the correct percentage.

12. Issues during the sequencing run

Below is a list of the most commonly encountered issues, with some suggested causes and solutions.

We also have an FAQ section available on the Nanopore Community Support section.

If you have tried our suggested solutions and the issue still persists, please contact Technical Support via email (support@nanoporetech.com) or via LiveChat in the Nanopore Community.

Fewer pores at the start of sequencing than after Flow Cell Check

Observation Possible cause Comments and actions
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check An air bubble was introduced into the nanopore array After the Flow Cell Check it is essential to remove any air bubbles near the priming port before priming the flow cell. If not removed, the air bubble can travel to the nanopore array and irreversibly damage the nanopores that have been exposed to air. The best practice to prevent this from happening is demonstrated in this video.
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check The flow cell is not correctly inserted into the device Stop the sequencing run, remove the flow cell from the sequencing device and insert it again, checking that the flow cell is firmly seated in the device and that it has reached the target temperature. If applicable, try a different position on the device (GridION/PromethION).
MinKNOW reported a lower number of pores at the start of sequencing than the number reported by the Flow Cell Check Contaminations in the library damaged or blocked the pores The pore count during the Flow Cell Check is performed using the QC DNA molecules present in the flow cell storage buffer. At the start of sequencing, the library itself is used to estimate the number of active pores. Because of this, variability of about 10% in the number of pores is expected. A significantly lower pore count reported at the start of sequencing can be due to contaminants in the library that have damaged the membranes or blocked the pores. Alternative DNA/RNA extraction or purification methods may be needed to improve the purity of the input material. The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

MinKNOW script failed

Observation Possible cause Comments and actions
MinKNOW shows "Script failed"
Restart the computer and then restart MinKNOW. If the issue persists, please collect the MinKNOW log files and contact Technical Support. If you do not have another sequencing device available, we recommend storing the flow cell and the loaded library at 4°C and contact Technical Support for further storage guidance.

Pore occupancy below 40%

Observation Possible cause Comments and actions
Pore occupancy <40% Not enough library was loaded on the flow cell For the human genome sequencing protocols, 200-300 ng of good quality library should be loaded on to an R10.4.1 flow cell to keep pore occupancy high.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and sequencing adapters did not ligate to the DNA Make sure to use the NEBNext Quick Ligation Module (E6056) and Oxford Nanopore Technologies Ligation Buffer (LNB, provided in the SQK-LSK114 kit) at the sequencing adapter ligation step, and use the correct amount of each reagent. A Lambda control library can be prepared to test the integrity of the third-party reagents.
Pore occupancy close to 0 The Ligation Sequencing Kit was used, and ethanol was used instead of LFB or SFB at the wash step after sequencing adapter ligation Ethanol can denature the motor protein on the sequencing adapters. Make sure the LFB or SFB buffer was used after ligation of sequencing adapters.
Pore occupancy close to 0 No tether on the flow cell Tethers are adding during flow cell priming (FCT tube). Make sure FCT was added to FCF before priming.

Shorter than expected read length

Observation Possible cause Comments and actions
Shorter than expected read length Unwanted fragmentation of DNA sample Read length reflects input DNA fragment length. Input DNA can be fragmented during extraction and library prep.

1. Please review the Extraction Methods in the Nanopore Community for best practice for extraction.

2. Visualise the input DNA fragment length distribution on an agarose gel before proceeding to the library prep. DNA gel2 In the image above, Sample 1 is of high molecular weight, whereas Sample 2 has been fragmented.

3. During library prep, avoid pipetting and vortexing when mixing reagents. Flicking or inverting the tube is sufficient.

Large proportion of unavailable pores

Observation Possible cause Comments and actions
Large proportion of unavailable pores (shown as blue in the channels panel and pore activity plot)

image2022-3-25 10-43-25 The pore activity plot above shows an increasing proportion of "unavailable" pores over time.		 Contaminants are present in the sample Some contaminants can be cleared from the pores by the unblocking function built into MinKNOW. If this is successful, the pore status will change to "sequencing pore". If the portion of unavailable pores stays large or increases:

1. A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be performed, or
2. Run several cycles of PCR to try and dilute any contaminants that may be causing problems.

Large proportion of inactive pores

Observation Possible cause Comments and actions
Large proportion of inactive/unavailable pores (shown as light blue in the channels panel and pore activity plot. Pores or membranes are irreversibly damaged) Air bubbles have been introduced into the flow cell Air bubbles introduced through flow cell priming and library loading can irreversibly damage the pores. Watch the Priming and loading your flow cell video for best practice
Large proportion of inactive/unavailable pores Certain compounds co-purified with DNA Known compounds, include polysaccharides.

1. Clean-up using the QIAGEN PowerClean Pro kit.
2. Perform a whole genome amplification with the original gDNA sample using the QIAGEN REPLI-g kit.
Large proportion of inactive/unavailable pores Contaminants are present in the sample The effects of contaminants are shown in the Contaminants Know-how piece. Please try an alternative extraction method that does not result in contaminant carryover.

Temperature fluctuation

Observation Possible cause Comments and actions
Temperature fluctuation The flow cell has lost contact with the device Check that there is a heat pad covering the metal plate on the back of the flow cell. Re-insert the flow cell and press it down to make sure the connector pins are firmly in contact with the device. If the problem persists, please contact Technical Services.

Failed to reach target temperature

Observation Possible cause Comments and actions
MinKNOW shows "Failed to reach target temperature" The instrument was placed in a location that is colder than normal room temperature, or a location with poor ventilation (which leads to the flow cells overheating) MinKNOW has a default timeframe for the flow cell to reach the target temperature. Once the timeframe is exceeded, an error message will appear and the sequencing experiment will continue. However, sequencing at an incorrect temperature may lead to a decrease in throughput and lower q-scores. Please adjust the location of the sequencing device to ensure that it is placed at room temperature with good ventilation, then re-start the process in MinKNOW. Please refer to this link for more information on MinION temperature control.

Last updated: 8/23/2024

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