This protocol describes a nanopore sequencing method for low-pass whole genome sequencing to enable methylation-based classification of four tumour samples per PromethION Flow Cell. It is designed to generate sufficient genome-wide coverage for accurate methylation classification and arm-level copy number variation (CNV) while maximising sample throughput.

All Oxford Nanopore Technologies supplied reagents required for this protocol are included in the Multiplex Methylation Whole Genome Sequencing bundle available in 24-sample format. This bundle includes the Native Barcoding Kit and PromethION Flow Cells, and is the standard configuration for implementing this workflow.

Genomic DNA is extracted from fresh or frozen tumour tissue (or provided as purified DNA), and sequencing libraries are prepared using the Native Barcoding Kit (SQK-NBD114.24), allowing multiplexing of four samples per run on a flow cell. Barcoding enables robust sample tracking and demultiplexing to ensure accurate assignment of sequencing reads to each tumour sample.

Whole genome sequencing is performed for 16 hours, generating low-pass whole genome coverage suitable for methylation profiling and CNV calling. Analysis can be performed using the Somatic analysis bioinformatics guide which provides an overview of methods using third-party tools.

Overview of steps in the workflow

Prepare for your experiment

You will need to:

• Extract DNA from your input sample (e.g. tumour tissue).
• Ensure you have your sequencing kit, the correct equipment, and third-party reagents.
• Download the software for acquiring and analysing your data.
• Ensure your analysis workstation or compute environment meets the hardware and software requirements of your chosen third-party analysis tools.
• Check your flow cell to ensure it has sufficient pores for a good sequencing run.

Sample preparation, library preparation, and sequencing

Step in workflow	Process	Time	Stop option
Sample preparation	Extract gDNA from sample and quantify. Fragment the gDNA and quantify. Check the length, quantity and purity of your extracted material. The quality checks performed during the protocol are recommended to ensure experimental success.	Dependent on extraction kit used ~15 minutes for shearing	At this stage, the extracted gDNA or fragmented gDNA can be stored at –20°C for later use.
DNA repair and end-prep	Repair the gDNA, and prepare the DNA ends for barcode and adapter attachment.	60 minutes	4°C overnight
Native barcode and adapter ligation	Ligate the native barcodes and sequencing adapters to the DNA ends.	90 minutes	4°C overnight
Priming and loading the flow cell	Prime the flow cell, and load your DNA library into the flow cell.	10 minutes
Sequencing	Sequence your library using your sequencing device.	16 hours
Data analysis	Refer to section 11, Downstream analysis and the Somatic analysis bioinformatics guide.

Oxford Nanopore has tested and recommends the extraction kits for tumour tissue samples listed below, as well as the DNA input requirement.

	Tumour tissue (fresh or frozen)
Kits	QIAamp Fast DNA Tissue Kit (Qiagen, 51404) Puregene Tissue Kit (Qiagen, 158063) MagAttract HMW DNA Kit (Qiagen, 67563)
DNA input per sample	250 ng Up to 1 µg input is recommended where possible to ensure robust and consistent performance.

Note: You will need to refer to the relevant kit handbook and protocol to determine which equipment and additional reagents are required for your extraction.

All Oxford Nanopore Technologies reagents required for this protocol are included in the Multiplex Methylation Whole Genome Sequencing bundle, available in the Nanopore Store in 24-sample format.

This bundle includes the Native Barcoding Kit and PromethION Flow Cells, and is the standard configuration for implementing this workflow.

For more information on specific kit components found within this bundle, please refer to the relevant product found in the Nanopore Store page:

• Multiplex Methylation Whole Genome Sequencing bundle 24: for 24 samples

• 250 ng of sheared gDNA per sample

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.

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.

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

We highly recommend that you check the number of pores in your flow cell prior to starting a sequencing experiment, within the warranty period. Flow cells should be removed from refrigeration and their packaging and allowed to come to room temperature for 30 minutes before they are used.

To perform the flow cell check, please follow the instructions in the Flow Cell Check document. As part of your flow cell check, you will see the number of pores in your new flow cell. The warranty on your flow cell covers a minimum pore count of 5,000 pores for a PromethION Flow Cell. You can find more information about the warranty period, stability and storage conditions of flow cells on the Oxford Nanopore Store. If you have any concerns or questions, please contact Oxford Nanopore.

Oxford Nanopore will replace any unused PromethION Flow Cell with fewer than 5,000 pores, when the result is reported within two days of performing the flow cell check, and when the storage recommendations have been followed.

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 PromethION 24 (P24) or PromethION 2 Integrated (P2i) device.

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

• PromethION 24: Starting a sequencing run on PromethION 24
• PromethION 2 Integrated: Starting a sequencing run on PromethION 2 Integrated

Below are the recommended sequencing parameters for MinKNOW.

Open the MinKNOW software UI via your desktop application.

In the MinKNOW UI, choose Start sequencing.

Follow the sequencing run set up on-screen using the settings defined below.

• Positions

  • Flow cell position: [user defined]
  • Experiment name: [user defined]
  • Flow cell type: FLO-PRO114M
  • Sample ID: [user defined]

• Kit selection

  • Kit selection: Native Barcoding Sequencing Kit (SQK-NBD114)

• Run configuration

  • Sequencing and analysis
  • Basecalling: On [default]
  • Modified bases: On (5hmC and 5mC in CG contexts)
  • Model: High accuracy basecalling (HAC)
  • Barcoding: On [default]

• Alignment: On

  • Reference for alignment: GRCh38_GIABv3

• Adaptive sampling: Off

• Data targets

  • Run limit: Stop when sequencing reaches 16 h

• Output Settings

  • Saved output file location: remote location for third-party methylation classification analysis or local directory [user defined]

• Output format

  • POD5: Off
    Note: Enable POD5 output if downstream re-basecalling is anticipated
  • FASTQ: Off
  • BAM: On [default]
  • Based on: Time elapsed
  • Frequency: Every 1 hour [default]

Methylation classification using third-party tools can be performed after 2 hours of sequencing, or once approximately 0.75 Gb of pass data has been generated per sample.

Methylation calls are contained within the BAM files generated by MinKNOW, which are saved in the run output directory:

/data/{experiment_group}/{sample_id}/{start_time}_{device_id}_{flow_cell_id}_{short_run_id}/bam_pass/

Example:

/data/TMP_Verification/VQL/20260514_1430_P2S-01234-_PAA12345_e05460c6/bam_pass/

CNV calling can be performed after 16 hours of sequencing, when each sample is expected to generate sufficient data, with target outputs of >6 Gb (>2x) of PASS data per barcode.

The Somatic analysis bioinformatics guide provides an overview of methods for the analysis.

Due to the multiplexing of samples, we do not recommend reusing the flow cells used in this method.

Reusing these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.

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.
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.

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 how to load a PromethION Flow Cell.
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.
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 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	The minimum recommended library loaded here is ~100 ng as DNA is sheared. Check Qubit measurements and perform fragment length QC to troubleshoot.
Pore occupancy close to 0	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 added 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. 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: A nuclease flush using the Flow Cell Wash Kit (EXP-WSH004) can be performed.

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 how to load a PromethION Flow Cell video for best practice.
Large proportion of inactive/unavailable pores	Certain compounds co-purified with DNA	Known compounds, include polysaccharides. Clean-up using the QIAGEN PowerClean Pro 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.