This protocol describes the process for library preparation and sequencing of one single sample per PromethION Flow Cell, using the Native Barcoding Kit and the Adaptive Sampling target enrichment function in MinKNOW. All Oxford Nanopore Technologies supplied reagents required for this protocol are included in the Singleplex Adaptive Sampling bundles available in 6-sample or 18-sample formats. These bundles include the Native Barcoding Kit, the auxiliary kits and all nanopore sequencing components, and are the standard configuration for implementing this workflow.

You can use an Oxford Nanopore ready-to-use, research-use-only digital panel as your BED-file-defined panel or create a custom digital panel using the Adaptive Sampling, Digital Panel Designer. For information on our ready-to-use panels or to create a custom panel, refer to the Adaptive Sampling technical guide - Digital Panel Designer.

We recommend sequencing for 72 hours to maximise depth across target regions. The resulting on-target coverage enables confident detection of somatic single nucleotide variants (SNVs), copy number variations (CNVs), indels, and structural variants (SVs), alongside whole-genome methylation profiling from the same dataset. The Somatic analysis bioinformatics guide provides an overview of methods for downstream analysis.

This protocol is recommended for tumour-only somatic oncology applications and other use cases requiring both methylation data and high on-target coverage (>75x).

Oxford Nanopore digital panel specification

Ready-to-use digital panels:

	Hereditary Cancer	Pan-Caner	Pan-Haematological Cancer
Enriched genes	258 inherited cancer risk genes	723 cancer associated genes	144 haematological malignancies associated genes
Validated Adaptive Sampling protocols	Multiplex Adaptive Sampling	Ultra-rapid singleplex Adaptive Sampling Singleplex Adaptive Sampling	Singleplex Adaptive Sampling
Input amount†	1 µg	Ultra-rapid singleplex: 1 µg Singleplex: 500 ng to 1 µg*	1 µg
Input sample type	Blood	Solid tumour tissue	Blood Bone marrow
Recommended extraction kits	Genomic DNA extraction from human blood using the Qiagen Puregene kit‡	1. QIAamp Fast DNA Tissue Kit (Qiagen, 51404) Recommended for Ultra-rapid singleplex Adaptive Sampling 2. Puregene Tissue Kit (Qiagen, 158063) 3. MagAttract HMW DNA Kit (Qiagen, 67563)	1. Genomic DNA extraction from human blood using the Qiagen Puregene kit‡ 2. QIAamp DSP DNA Mini Kit (Qiagen, 61304) 3. GenFind V3 Genomic DNA Extraction Kit (Beckman Coulter, C34880)
QC info (Femto Pulse)	Extracted gDNA samples may be assessed on the Femto Pulse System to visualise the unsheared/sheared gDNA profiles. For example profiles refer to the Input DNA section.	Extracted gDNA samples may be assessed on the Femto Pulse System to visualise the unsheared/sheared gDNA profiles. For example profiles refer to the DNA QC section. For the Ultra-rapid singleplex AS protocol, perform gDNA quality control assessment after completion of the sequencing run using aliquots reserved during the relevant sample preparation steps.	Extracted gDNA samples may be assessed on the Femto Pulse System to visualise the unsheared/sheared gDNA profiles. For example profiles refer to the DNA QC section.
Shearing conditions (speed and time)	Three centrifugation steps at ~3,381 RCF for 1 minute each. Refer to the corresponding protocol step for further details.	Two centrifugation steps at ~3,381 RCF for 30 seconds each. Refer to the corresponding protocol step for further details.	Two centrifugation steps at ~4,868 RCF for 1 minute each. Refer to the corresponding protocol step for further details.
Reference genomes	GRCh38_GIABv3	GRCh38_GIABv3	GCA_000001405.15_GRCh38_no_alt_analysis_set.fna
Enrichment BED file (links to the BED files can be found on the Adaptive Sampling page)	ONT_AS_HCP_GRCh38_enrichment_v1.bed	ONT_AS_PanCancer_GRCh38_enrichment_v1.bed	ONT_AS_PanHaem_GRCh38_enrichment_v1.bed

*A lower input limit of 500 ng can be used; however 1 µg is recommended whenever possible to improve pore occupancy.

^†A maximum of 2 µg input can be used to maximise pore occupancy.

^‡This Oxford Nanopore adapted extraction method uses the Puregene Blood Kit (Qiagen, 158023). It has gentler mixing conditions and resuspends the final DNA at 50°C rather than 65°C.

Prepare for your experiment

You will need to:

• Extract DNA from your input sample (fresh or frozen tumour tissue, whole blood or bone marrow).
• 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.	72 hours
Data analysis	Refer to section 11, Downstream analysis and to the Somatic analysis bioinformatics guide.

Los reactivos de Oxford Nanopore Technologies necesarios en este protocolo están incluidos en los paquetes de Muestreo adaptativo con multiplexación, de venta en la tienda Nanopore en formato de 18 o 72 muestras.

Esos paquetes incluyen el kit Native Barcoding junto con el resto de elementos de secuenciación que constituyen la configuración de referencia en este flujo de trabajo.

Si desea más información sobre elementos concretos del kit incluidos en los paquetes, consulte los productos correspondientes en la tienda Nanopore:

• Paquete Muestreo adaptativo con multiplexación 18 (18 muestras)
• Paquete Muestreo adaptativo con multiplexación 72 (72 muestras)

• 1 μg of sheared gDNA

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.

Make sure that the dark grey heat pad on the underside of the flow cell is not torn or deformed.

For the PromethION 24:

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.

For the PromethION 2 Integrated:

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

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

3. Press the flow cell firmly into the loader until a click is heard.

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

We recommend basecalling with the super accurate (SUP) 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

Note: DNA libraries prepared from blood and bone marrow samples have been sequenced using the PromethION 2 Integrated only.

We strongly recommend using MinKNOW version 26.01.21 LTS or newer for optimal results.

Make sure you have the correct Adaptive Sampling files for MinKNOW (reference genome FASTA file and target region BED file) available on your device for data acquisition and analysis.

For ready-to-use digital panels, refer to section 2.

If you require assistance ensuring you are on the correct version of MinKNOW, please contact your Oxford Nanopore Technologies representative.

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-NBD114.24)

• Run configuration

  • Sequencing and analysis
  • Basecalling: On [default]
  • Modified bases: On (5hmC and 5mC in CG contexts)
  • Model: Super accurate basecalling: (SUP)
  • Barcoding: On [default]

Note: If running multiple flow cells simultaneously, there may be a basecalling lag, so account for the extra time needed for catch-up.

• Alignment: On

  • Reference for alignment: see recommendations below
  • Optional file enumerating regions: add BED file of choice

• Adaptive sampling: On

  • Enrich selected
  • Basecall on-target reads only: Off [default]
  • Reference for alignment: see recommendations below
  • File enumerating regions: add BED file of choice

• Data targets

  • Run limit: Stop when sequencing reaches 72 h [default]

• Output Settings

  • Saved output file location: remote location for third-party methylation classification analysis

• 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 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/TumourMethylationProfiling/VQL/20260514_1430_P2S-01234-_PAA12345_e05460c6/bam_pass/

If proceeding to variant calling, we recommend this is performed after 72 hours of sequencing, to achieve maximum coverage.

Refer to the Somatic analysis bioinformatics guide for an overview of possible methods for the analysis.

Due to the extended sequencing time, 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	For this protocol, 50 to 65 fmol 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	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 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.