Ligation sequencing gDNA - reduced representation methylation sequencing (RRMS) of human samples (SQK-LSK110) (RRMS_9164_v110_revD_30May2022)
GridION: Protocol
V RRMS_9164_v110_revD_30May2022
FOR RESEARCH USE ONLY
Contents
Introduction to the protocol
- 1. Overview of the protocol
- 2. Equipment and consumables
- 3. .bed file
- 4. Computer requirements and software
Sample and library preparation
- 5. DNA extraction
- 6. DNA repair and end-prep
- 7. Adapter ligation and clean-up
- 8. Priming and loading the SpotON flow cell for GridION
Sequencing and data analysis
Troubleshooting
1. Overview of the protocol
This is a Legacy product
This kit is available on the Legacy page of the store. We are in the process of gathering data to support the upgrade of this protocol to our latest chemistry. Further information regarding protocol upgrades will be provided on the Community as soon as they are available over the next few months. For further information on please see the product update page.
Reduced representation methylation sequencing (RRMS)
Nanopore sequencing enables direct detection of methylated cytosines (e.g., at CpG sites), without the need for bisulphite conversion. CpG sites frequently occur in high density clusters called CpG islands (CGI) and >60% of human genes have their promoters embedded within CGIs.
Changes in methylation patterns within promoters is associated with changes in gene expression and disease states such as cancer: exploring methylation differences between tumour samples and normal samples can help to uncover mechanisms associated with tumour formation and development.
Adaptive sampling (AS) offers a fast, flexible and precise method to enrich for regions of interest (e.g. CGIs) by depleting off-target regions during sequencing itself with no requirement for upfront sample manipulation. Here we introduce Reduced Representation Methylation Sequencing (RRMS): Oxford Nanopore’s methylation detection is combined with AS to target 310 Mb of the human genome which are highly enriched for CpGs including ~28,000 CpG islands, ~50,600 shores and ~42,700 shelves as well as ~21,600 promotor regions.
To benchmark, we performed RRMS on five replicates of a metastatic melanoma cell line and its normal pair for a male individual (COLO829/COLO829_BL) and a triple negative breast cancer cell-line pair (HCC1395/HCC1935_BL). Each sample was run on a single MinION flow cell: RRMS resulted in high-confidence methylation calls (>10 overlapping reads) for 7.3-8.5 million CpGs per sample.
For more background information about designing an adaptive sampling experiment, please refer to the Adaptive sampling best practice document.
Introduction to the DNA extraction and Ligation Sequencing protocol for RRMS
This protocol describes how to carry out DNA extraction and reduced representation methylation sequencing (RRMS) of human samples using the Ligation Sequencing Kit (SQK-LSK110) and the Adaptive Sampling feature in MinKNOW.
Steps in the sequencing workflow:
Prepare for your experiment
You will need to:
- Extract your DNA, fragment it using the Covaris g-TUBE, and check its length, quantity and purity. The quality checks performed during the protocol are essential in ensuring experimental success.
- Ensure you have your sequencing kit, the correct equipment and third-party reagents
- Download the software for acquiring and analysing your data
- Ensure that you have the correct .bed file for Adaptive Sampling
- Check your flow cell to ensure it has enough pores for a good sequencing run
Library preparation
You will need to:
- Repair the DNA, and prepare the DNA ends for adapter attachment
- Attach sequencing adapters supplied in the kit to the DNA ends
- Prime the flow cell, and load your DNA library into the flow cell
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. While configuring the run, turn on the Adaptive Sampling setting and import a pre-prepared .bed file with your regions of interest, along with a FASTA reference file.
- Sequence the sample for a total of 96 hours, with two flow cell washes when the available pore count drops to around 40% of the starting pore count (typically after ~30 hours and the second time after ~64 hours).
- Use the Guppy protocol to call modified bases, and then the commands recommended at the end of this protocol to aggregate the modified bases and perform CpG island annotation.
Compatibility of this protocol
This protocol should only be used in combination with:
- Ligation Sequencing Kit (SQK-LSK110)
- FLO-MIN106 (R9.4.1) flow cells
- Flow Cell Wash Kit (EXP-WSH004)
2. Equipment and consumables
Material
- 5 x 10⁶ cells (e.g. cell culture or tissue sample)
- Ligation Sequencing Kit (SQK-LSK110)
- Flow Cell Wash Kit (EXP-WSH004)
Consumibles
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (NEB E7180S or E7180L) (módulo de acompañamiento NEBNext de secuenciación por ligación para Oxford Nanopore Technologies®) Como alternativa, se pueden utilizar los siguientes productos de NEBNext®:
- NEBNext FFPE Repair Mix (NEB M6630) (mezcla de reparación de ADN)
- NEBNext Ultra II End Repair/dA-tailing Module (NEB E7546) (Módulo de reparación de extremos/Adición de dA)
- NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
- Freshly prepared 70% ethanol in nuclease-free water
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Tubos de PCR de pared fina (0,2 ml)
Instrumental
- Mezclador Hula (mezclador giratorio suave)
- Separador magnético, adecuado para tubos Eppendorf de 1,5 ml
- Microcentrífuga
- Mezclador vórtex
- Termociclador
- Pipeta y puntas P1000
- Pipeta y puntas P200
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
- Pipeta y puntas P2
- Cubeta con hielo
- Temporizador
Equipo opcional
- Bioanalizador Agilent (o equivalente)
- Fluorímetro Qubit (o equivalente para el control de calidad)
- Centrifugadora Eppendorf 5424 (o equivalente)
For this protocol, you will need 5 x 10⁶ cells or another human sample, e.g. blood or tissue. After performing DNA extraction and DNA fragmentation, you will need 2 µg genomic DNA to take forward into the library preparation.
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.
NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing
For customers new to nanopore sequencing, we recommend buying the NEBNext® Companion Module for Oxford Nanopore Technologies® Ligation Sequencing (catalogue number E7180S or E7180L), which contains all the NEB reagents needed for use with the Ligation Sequencing Kit.
Please note, for our amplicon protocols, NEBNext FFPE DNA Repair Mix and NEBNext FFPE DNA Repair Buffer are not required.
Ligation Sequencing Kit (SQK-LSK110) contents
| Name | Acronym | Cap colour | No. of vials | Fill volume per vial (µl) |
|---|---|---|---|---|
| DNA CS | DCS | Yellow | 1 | 35 |
| Adapter Mix F | AMX-F | Green | 1 | 40 |
| Ligation Buffer | LNB | Clear | 1 | 200 |
| L Fragment Buffer | LFB | White cap, orange stripe on label | 2 | 1,800 |
| S Fragment Buffer | SFB | Grey | 2 | 1,800 |
| Sequencing Buffer II | SBII | Red | 1 | 500 |
| Elution Buffer | EB | Black | 1 | 200 |
| Loading Beads II | LBII | Pink | 1 | 360 |
| Loading Solution | LS | White cap, pink sticker on label | 1 | 360 |
| Flush Buffer | FB | Blue | 6 | 1,170 |
| Flush Tether | FLT | Purple | 1 | 200 |
3. .bed file
Download the .bed file from the Adaptive Sampling catalogue.
The Adaptive Sampling catalogue provides a way for both the Oxford Nanopore team and Community members to share .bed files with genomic target regions used for Adaptive Sampling experiments. The .bed files along with a reference genome can be uploaded into MinKNOW.
For RRMS experiments, download the Reduced representation methylation sequencing (RRMS) file.
4. Computer requirements and software
GridION IT requirements
The GridION device contains all the hardware required to control up to five flow cells and acquire the data. The device is further enhanced with high performance GPU technology for real-time basecalling. Read more in the GridION IT requirements document.
Software for nanopore sequencing
MinKNOW
The MinKNOW software controls the nanopore sequencing device, collects sequencing data and basecalls in real time. You will be using MinKNOW for every sequencing experiment to sequence, basecall and demultiplex if your samples were barcoded.
For instructions on how to run the MinKNOW software, please refer to the MinKNOW protocol.
EPI2ME (optional)
The EPI2ME cloud-based platform performs further analysis of basecalled data, for example alignment to the Lambda genome, barcoding, or taxonomic classification. You will use the EPI2ME platform only if you would like further analysis of your data post-basecalling.
For instructions on how to create an EPI2ME account and install the EPI2ME Desktop Agent, please refer to this link.
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 |
5. DNA extraction
DNA extraction
Extract DNA from your sample using one of our recommended extraction protocols. For the benchmarking of this method, the Oxford Nanopore team extracted DNA from ~5 million cells using the protocol: Human cell line DNA – QIAGEN Puregene Cell Kit. We also offer multiple mammalian sample extraction protocols, which you can use for other sample types.
DNA fragmentation
The DNA was sheared using the protocol for Covaris g-TUBE fragmentation, with the following modifications:
- The input was 8 μg of DNA in 150 μl.
- The DNA was fragmented in the g-TUBE at 11,000 rpm in 30 sec pulses. The resulting fragment length should be ~6-7 kb.
6. DNA repair and end-prep
Material
- gDNA in 47 µl nuclease-free water
- DNA Control Sample (DCS) (muestra de control)
Consumibles
- Tubos de PCR de pared fina (0,2 ml)
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
- NEBNext FFPE DNA Repair Mix (NEB M6630)
- NEBNext Ultra II End repair/dA-tailing Module (NEB E7546)
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Freshly prepared 70% ethanol in nuclease-free water
Instrumental
- Pipeta y puntas P1000
- Pipeta y puntas P100
- Pipeta y puntas P10
- Termociclador
- Microcentrífuga
- Mezclador Hula (mezclador giratorio suave)
- Gradilla magnética
- Cubeta con hielo
Thaw DNA Control Sample (DCS) at room temperature, spin down, mix by pipetting, and place on ice.
Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.
For optimal performance, NEB recommend the following:
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.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to room temperature and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate.
Note: It is important the buffers are mixed well by vortexing.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 2 μg of the fragmented DNA into a 1.5 ml Eppendorf DNA LoBind tube.
- Adjust the volume to 47 μl with nuclease-free water
- Mix thoroughly by flicking the tube
- Spin down briefly in a microfuge
In a 0.2 ml thin-walled PCR tube, mix the following:
Between each addition, pipette mix 10-20 times.
| Reagent | Volume |
|---|---|
| DNA from the previous step | 47 µl |
| DNA CS (optional) | 1 µl |
| NEBNext FFPE DNA Repair Buffer | 3.5 µl |
| NEBNext FFPE DNA Repair Mix | 2 µl |
| Ultra II End-prep Reaction Buffer | 3.5 µl |
| Ultra II End-prep Enzyme Mix | 3 µl |
| Total | 60 µl |
Ensure the components are thoroughly mixed by pipetting, and spin down.
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
AMPure XP bead clean-up
It is recommended that the repaired/end-prepped DNA sample is subjected to the following clean-up with AMPure XP beads. This clean-up can be omitted for simplicity and to reduce library preparation time. However, it has been observed that omission of this clean-up can: reduce subsequent adapter ligation efficiency, increase the prevalence of chimeric reads, and lead to an increase in pores being unavailable for sequencing. If omitting the clean-up step, proceed to the next section.
Resuspend the AMPure XP beads by vortexing.
Transfer the DNA sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
Add 60 µl of resuspended AMPure XP beads 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 500 μl of fresh 70% 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 70% 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.
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.
7. Adapter ligation and clean-up
Material
- Adapter Mix F (AMX F)
- Ligation Buffer (LNB) (tampón de ligación) del kit Ligation Sequencing Kit
- Long Fragment Buffer (LFB) (tampón para fragmentos largos)
- Short Fragment Buffer (SFB) (tampón para fragmentos cortos)
- Elution Buffer (EB) (tampón de elución) del kit de Oxford Nanopore
Consumibles
- NEBNext Quick Ligation Module (NEB E6056) (Módulo de ligación rápida)
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
Instrumental
- Gradilla magnética
- Microcentrífuga
- Mezclador vórtex
- Pipeta y puntas P1000
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
Although the recommended 3rd party ligase is supplied with its own buffer, the ligation efficiency of Adapter Mix F (AMX-F) is higher when using Ligation Buffer supplied within the Ligation Sequencing Kit.
Spin down the Adapter Mix F (AMX-F) and Quick T4 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.
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)
To enrich for DNA fragments of 3 kb or longer, thaw one tube of Long Fragment Buffer (LFB) at room temperature, mix by vortexing, spin down and place on ice.
To retain DNA fragments of all sizes, thaw one tube of Short Fragment Buffer (SFB) at room temperature, mix by vortexing, 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 Buffer (LNB) | 25 µl |
| NEBNext Quick T4 DNA Ligase | 10 µl |
| Adapter Mix F (AMX-F) | 5 µl |
| Total | 100 µl |
Ensure the components are thoroughly mixed by pipetting, and spin down.
Incubate the reaction for 10 minutes at room temperature.
If you have omitted the AMPure purification step after DNA repair and end-prep, do not incubate the reaction for longer than 10 minutes.
Resuspend the AMPure XP beads by vortexing.
Add 40 µl of resuspended AMPure XP beads 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 either 250 μl Long Fragment Buffer (LFB) or 250 μl 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 room temperature. 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.
Dispose of the pelleted beads
Quantify 1 µl of eluted sample using a Qubit fluorometer.
We recommend loading 150 ng of the final prepared library onto the flow cell.
Loading more than the maximal recommended amount of DNA can have a detrimental effect on output as higher quantities of DNA results in a larger number of ligated DNA ends with loaded motor protein. This depletes fuel in the Sequencing Buffer, regardless of whether or not the DNA fragments are being sequenced. This leads to fuel depletion and speed drop-off early in the sequencing run. Dilute the libraries in Elution Buffer if required.
The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.
Library storage recommendations
We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, reloading 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. For further information, please refer to the DNA library stability Know-How document.
Sequencing and flow cell washes
Sequence the sample for a total of 96 hours, with two flow cell washes. After ~30 hours, or when the pore count drops to 40-50% of the initial number at the start of the experiment, pause the run and wash the flow cell using the Flow Cell Wash Kit. Load another 150 ng of library and sequence for another ~23 hours. After this, repeat the flow cell wash for the second time, load another 150 ng of library and sequence for the remaining ~43 hours.
Note: To avoid pore numbers falling too low before performing the flow cell wash, it may be necessary to pause the experiment overnight.
8. Priming and loading the SpotON flow cell for GridION
Material
- Loading Solution (LS)
- Sequencing Buffer II (SBII)
- Loading Beads II (LBII)
- Flush Buffer (FB)
- Flush Tether (FLT)
Consumibles
- Tubos de 1,5 ml Eppendorf DNA LoBind
- Agua sin nucleasas (p. ej., ThermoFisher AM9937)
Instrumental
- GridION device
- Pipeta y puntas P1000
- Pipeta y puntas P100
- Pipeta y puntas P20
- Pipeta y puntas P10
Priming and loading a flow cell
We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.
Using the Loading Solution
We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water. Note: some customers have noticed that viscous libraries can be loaded more easily when not using Loading Beads II.
Please note that the Sequencing Tether (SQT) tube will NOT be used in this protocol.
Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature before mixing the reagents by vortexing and spin down at room temperature.
To prepare the flow cell priming mix, add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.
Slide open the GridION lid and insert the flow cell.
Press down firmly on the flow cell to ensure correct thermal and electrical contact.
Complete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
See the flow cell check instructions in the MinKNOW protocol for more information.
Slide the flow cell priming port cover clockwise to open the priming port.
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 Loading Beads II (LBII) by pipetting.
The Loading Beads II (LBII) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
In a new tube, prepare the library for loading as follows:
| Reagent | Volume per flow cell |
|---|---|
| Sequencing Buffer II (SBII) | 37.5 µl |
| Loading Beads II (LBII), mixed immediately before use, or Loading Solution (LS), if using | 25.5 µl |
| DNA library | 12 µl |
| Total | 75 µl |
Note: Load the library onto the flow cell immediately after adding the Sequencing Buffer II (SBII) and Loading Beads II (LBII) because the fuel in the buffer will start to be consumed by the adapter.
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 μl 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, close the flow cell priming port and close the GridION lid.
9. Data acquisition and basecalling
Overview of nanopore data analysis
For a full overview of nanopore data analysis, which includes options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.
How to start sequencing
The sequencing device control, data acquisition, real-time basecalling, and barcode demultiplexing are carried out by the MinKNOW software. It is assumed you have already installed MinKNOW on your computer. Instructions for this can be found in the MinKNOW protocol.
Open the MinKNOW software using the desktop shortcut and log into the MinKNOW software using your Community credentials.
Select your connected device.
Sequencing set-up
Select Start sequencing. 
Select positions
Enter your Experiment name and sample ID as prompted. 
Kit selection
The kit selection tab will provide a dropdown of available kits. Check the SQK-LSK110 kit.
Run options
The run options tab provides variables for run time and starting voltage. Leave these at the default values.
Under Adaptive sampling, upload the .bed file and reference FASTA file.
Basecalling
Check Basecalling and select the Fast basecalling option in the dropdown menu.
Output
Select where you would like to save the data.
Review
Review your sequencing set-up and click Start.
10. Downstream analysis
Ensure you have installed the latest version of Guppy. To perform basecalling and methylation calling using Remora, open a terminal and enter the following commands:
guppy_basecaller \
-i {input_fast5s} -s {output_folder} \
-c dna_r9.4.1_450bps_modbases_5mc_cg_hac.cfg \
--align_ref {reference_fasta} \
--device auto \
--compress_fastq --bam_out --recursive \
--num_callers 5 --cpu_threads_per_caller 4
Concatenate all BAM files output by Guppy into one:
samtools cat {output_folder}/pass/*bam | \
samtools sort -@ 8 -o {out_bam} -
Index the merged BAM file:
samtools index -@ 8 {out_bam}
This will create a single sorted and indexed BAM file ({out_bam}) that contains canonical bases as well as per-read modifications and can be loaded into IGV. To visualise the per-read modification calls in IGV, load the BAM file and set "colour reads as" to "modifications".
This BAM file can be used to check the on-target coverage achieved during the Reduced Representation Methylation Sequencing (RRMS) run:
mosdepth -x -t 8 -n -b {target_bed} {prefix} {out_bam}
To create strand-specific and strand-aggregated methylation frequencies for all genomic positions (CpGs), run:
modbam2bed -m 5mC -e --cpg -t 8 -a 0.2 -b 0.8 \
--aggregate --prefix {prefix} \
{reference_fasta} \
{out_bam} > {out_mod_bed}
This will create two BEDMETHYL files: one will report methylation frequencies per genomic position and per strand, the second file will include the prefix specified and will report methylation frequencies by aggregating calls from the forward and reverse strand. The tool can be found in the following repository: https://github.com/epi2me-labs/modbam2bed
Filter reference CpG positions without canonical or modified calls (e.g. deletions from the reference) and genomic positions without calls from both strands:
cat {prefix}_cpg.acc.bed | csvtk filter2 -H -t \
-f '$11 != "nan" && $6 != "+" && $6 != "-"' > {out_mod_bed_agg_filt}
Convert the BEDMETHYL file to a TSV file that is compatible with the DMR tool DSS:
awk -v OFS='\t' 'BEGIN{print "chr","pos","N","X"}{print $1,$2,($12+$13),$13}' {out_mod_bed_agg_filt} > {out_mod_bed_agg_filt_DSS}
Convert the BEDMETHYL file to a BEDGRAPH file that will be used for obtaining the BIGWIG format useful for IGV visualisation:
awk -v OFS='\t' '{print $1,$2,$3,$11}' {out_mod_bed_agg_filt} | \
sort -k1,1 -k2,2n > {out_mod_bed_agg_filt_bedgraph}
bedGraphToBigWig {out_mod_bed_agg_filt_bedgraph} {reference_chrSize} {out_mod_bed_agg_filt_bigwig}
Repeat the above steps for all your samples.
For detection of differentially methylated regions use DSS as described here: https://bioconductor.org/packages/release/bioc/vignettes/DSS/inst/doc/DSS.html
Benchmarking results
For information about benchmarking the performance of RRMS, please see our RRMS performance document.
11. Issues during DNA/RNA 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 document. Please try an alternative extraction method that does not result in contaminant carryover. Consider performing an additional SPRI clean-up step. |
| Low RNA integrity (RNA integrity number <9.5 RIN, or the rRNA band is shown as a smear on the gel) | The RNA degraded during extraction | Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page. |
| RNA has a shorter than expected fragment length | The RNA degraded during extraction | Try a different RNA extraction method. For more info on RIN, please see the RNA Integrity Number document. Further information can be found in the DNA/RNA Handling page. We recommend working in an RNase-free environment, and to keep your lab equipment RNase-free when working with RNA. |
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.  |
| 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 | Ensure you load the recommended amount of good quality library in the relevant library prep protocol onto your flow cell. Please quantify the library before loading and calculate mols using tools like the Promega Biomath Calculator, choosing "dsDNA: µg to pmol" |
| 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 sequencing 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 (FLT/FCT tube). Make sure FLT/FCT was added to FB/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.  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)  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, typically associate with plant genomic DNA. 1. Please refer to the Plant leaf DNA extraction method. 2. Clean-up using the QIAGEN PowerClean Pro kit. 3. 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. |
Reduction in sequencing speed and q-score later into the run
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Reduction in sequencing speed and q-score later into the run | For Kit 9 chemistry (e.g. SQK-LSK109), fast fuel consumption is typically seen when the flow cell is overloaded with library (please see the appropriate protocol for your DNA library to see the recommendation). | Add more fuel to the flow cell by following the instructions in the MinKNOW protocol. In future experiments, load lower amounts of library to the flow cell. |
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. |
Guppy – no input .fast5 was found or basecalled
| Observation | Possible cause | Comments and actions |
|---|---|---|
| No input .fast5 was found or basecalled | input_path did not point to the .fast5 file location | The --input_path has to be followed by the full file path to the .fast5 files to be basecalled, and the location has to be accessible either locally or remotely through SSH. |
| No input .fast5 was found or basecalled | The .fast5 files were in a subfolder at the input_path location | To allow Guppy to look into subfolders, add the --recursive flag to the command |
Guppy – no Pass or Fail folders were generated after basecalling
| Observation | Possible cause | Comments and actions |
|---|---|---|
| No Pass or Fail folders were generated after basecalling | The --qscore_filtering flag was not included in the command | The --qscore_filtering flag enables filtering of reads into Pass and Fail folders inside the output folder, based on their strand q-score. When performing live basecalling in MinKNOW, a q-score of 7 (corresponding to a basecall accuracy of ~80%) is used to separate reads into Pass and Fail folders. |
Guppy – unusually slow processing on a GPU computer
| Observation | Possible cause | Comments and actions |
|---|---|---|
| Unusually slow processing on a GPU computer | The --device flag wasn't included in the command | The --device flag specifies a GPU device to use for accelerate basecalling. If not included in the command, GPU will not be used. GPUs are counted from zero. An example is --device cuda:0 cuda:1, when 2 GPUs are specified to use by the Guppy command. |
Last updated: 3/10/2023
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