This protocol describes the process for DNA extraction, library preparation, and analysis for the Hereditary Cancer Panel (HCP) for 3 samples 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 Hereditary Cancer Panel Bundle, available in 18 or 72 sample formats. This bundle includes the Native Barcoding Kit and all necessary sequencing components, and is the standard configuration for implementing this workflow.

Please note, after extraction if processing multiple samples, samples are to be processed in increments of three for library preparation.

Overview of steps in the workflow

Prepare for your experiment

You will need to:

• Extract your input sample (whole blood, DNA)
• 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, 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.	4 to 6 hours	At this stage, the extracted gDNA or fragmented gDNA can be stored at –20°Cfor later use.
DNA repair and end-prep	Repair the gDNA, and prepare the DNA ends for adapter attachment	60 minutes	4°C overnight
Native barcode ligation	Ligate the native barcodes to the DNA ends	70 minutes	4°C overnight
Sequencing adapter ligation and clean-up	Ligate sequencing adapters to the DNA ends	60 minutes	4°C for short-term storage or for repeated use, such as for reloading your flow cell –80°C for long-term storage
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	Analysis using HCP data bundle	120 minutes

Following the Hereditary cancer profiling (HCP) registration, a link will be provided by email to a file storage location containing the data bundle to initiate and analyse your sequencing run. This data bundle is specifically configured for this product and ensures consistent setup and compatibility. Instructions for installing the .2ME file in EPI2ME, including how to test it with demo data, are provided in the data analysis section of this protocol.

The data bundle contains:

• Hereditary Cancer adaptive sampling files for MinKNOW (reference genome .FASTA, target region .BED file) to be used during sequencing for target enrichment.

• Hereditary Cancer analysis workflow files for post-sequencing bioinformatic analysis in EPI2ME (.2ME).

• Example files

• If you have purchased the Hereditary Cancer bundle, and you have not received an email from the Oxford Nanopore CloudHub platform with a secure link to the required Data Bundle files, please contact our Support Team (support@nanoporetech.com) with your order ID and we will work to provide you with the required invitation.

All Oxford Nanopore Technologies reagents required for this protocol are included in the Hereditary Cancer Panel bundles, available in the Nanopore Store in 18-sample (HCP18) or 72-sample (HCP72) formats.

These bundle include the Native Barcoding Kit and all necessary sequencing components, and are the standard configuration for implementing this workflow.

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

• Hereditary Cancer Panel: 18 samples bundle (HCP18)
• Hereditary Cancer Panel: 72 samples bundle (HCP72)

Input requirements per sample for the extraction method:

• ≥ 1 ml of human blood in EDTA K2 vacuum tube

Input requirements per sample for the library preparation:

• ~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 Technologies.

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. This should be done within 12 weeks of purchasing for PromethION. 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
PromethION Flow Cell	5000

• If using fresh blood or blood stored at 4°C, proceed to DNA extraction.
• If using frozen blood, ensure your sample is thawed completely before proceeding.

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 P24 device.

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

• PromethION 24: "Starting a sequencing run with PromethION 24"

Below are the recommended sequencing parameters for MinKNOW.

We strongly recommend using MinKNOW version 25.03.7 for optimal results. Instructions for MinKNOW 25.03.7 rollback can be downloaded here.

Please ensure you have the correct BED Files available on your device for data acquisition and analysis.

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

Setting up your sequencing run in MinKNOW:

• Open the MinKNOW software UI via your desktop application.
• In the MinKNOW UI, you can choose either Start sequencing (option 1) or Start from a sample sheet (option 2).

• 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

Kit selection: Native Barcoding Sequencing Kit (SQK-NBD114.24)

Run configuration

Sequencing and analysis

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

Barcoding: On [default]

Alignment: On
Upload the reference genome file provided in the Hereditary Cancer Panel file pack (HCP_GRCh38_masked_v1.0.fasta) for alignment

Adaptive sampling: On
Enrich: On
Deplete: Off [default]
Barcode balancing: Off [default]
Basecall on-target reads only: Off [default]
Upload the reference genome file (HCP_GRCh38_masked_v1.0.fasta) and target region (HCP_GRCh38_masked_v1.0.bed) provided in the Hereditary Cancer Panel file pack for alignment

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]

Analysis workflow

Workflow: Off [default]

Output

Ensure enough space on your device: minimum of 0.8TB per flow cell.

Output format
.BAM: On [default]
.FASTQ: On [default]
Based on: Time elapsed [default]
Frequency: Every 10 minutes [default]
Barcoding: Split files by barcode: On [default]
FASTQ options: Compression: Off

Raw reads output: On [default]
.POD5: On [default]
.FAST5: Off [default]
Raw reads options: Split .POD5 files by barcode: Off [default]

Filtering: On [default]
Qscore: 9 [default]
Minimum read length: [default]
Max read length: [default]

Starting from a sample sheet (.csv format) enables assigning each barcode a name that will be carried through the analysis (alias column). The sample sheet defines the flow cell (flow_cell_product_code column) and kit (kit column) used. Additionally, a column for experiment (experiment_id column) and either slot number on the device (position_ID) or unique flow cell identifier (position_ID) is requiredIt is compulsory to add a column for either the sequencing slot number on the device( position_ID) or the flow cell unique identifier (flow_cell_ID).

For further information on sample sheet options, see the sample sheet upload section of our MinKNOW documentation.

• Select the sample sheet file from your file system. Please, ensure that your sample sheet name has a visible “.csv” extension for it to be recognised by MinKNOW.
• Follow the sequencing run set up on-screen using the settings defined below:

Positions

Flow cell position: [Defined in sample sheet]

Experiment name: [Defined in sample sheet]

Flow cell type: FLO-PRO114M

Sample ID: [Defined in sample sheet]

Kit

Kit selection: Native Barcoding Sequencing Kit (SQK-NBD114.24)

Run configuration

Sequencing and analysis

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

Barcoding: On [default]

Alignment: On
Upload the reference genome file provided in the Hereditary Cancer Panel file pack (HCP_GRCh38_masked_v1.0.fasta) for alignment

Adaptive sampling: On
Enrich: On
Deplete: Off [default]
Barcode balancing: Off [default]
Basecall on-target reads only: Off [default]
Upload the reference genome file (HCP_GRCh38_masked_v1.0.fasta) and target region (HCP_GRCh38_masked_v1.0.bed) provided in the Hereditary Cancer Panel file pack for alignment

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]

Analysis workflow

Workflow: Off [default]

Output

Ensure enough space on your device: minimum of 0.8TB per flow cell.

Output format
.BAM: On [default]
.FASTQ: On [default]
Based on: Time elapsed [default]
Frequency: Every 10 minutes [default]
Barcoding: Split files by barcode: On [default]
FASTQ options: Compression: Off

Raw reads output: On [default]
.POD5: On [default]
.FAST5: Off [default]
Raw reads options: Split .POD5 files by barcode: Off [default]

Filtering: On [default]
Qscore: 9 [default]
Minimum read length: [default]
Max read length: [default]

The sequencing output files (.bam) can be analysed using wf-hereditary-cancer, a bioinformatics workflow implemented in Nextflow that is part of Oxford Nanopore’s EPI2ME suite of analysis workflows. wf-hereditary-cancer can be run within the EPI2ME Desktop App. To run the analysis, you will need the workflow package (reference genome FASTA, target region BED files, and the workflow itself) distributed separately as a .2ME file.

Please note that we do not recommend sequencing and performing data analysis simultaneously on your device.

Installing EPI2ME and working directory setup

If not already installed on your device, you can download and install the EPI2ME Desktop App following these instructions. Once installed, click on ‘Settings’ on the main dashboard, and ensure the working directory is set to ‘/data/epi2melabs’ as follows. This will ensure that the files generated by the workflow during analysis do not fill up the system’s home directory:

The wf-hereditary-cancer analysis workflow is distributed as a .2ME file which is distributed via CloudHub. Please see the Before getting started: Data bundle and workflow setup section of the protocol for instructions on how to gain access to and download these files.

Running wf-hereditary cancer for the first time: installing the workflow 2ME package

If you are running wf-hereditary-cancer for the first time, follow these steps to install the workflow 2ME package:

1. Open the EPI2ME desktop application and select Launch from the menu on the left.

2. Select Import workflow on the right of the screen, and in the dropdown, select Import a 2ME file.

3. In the pop-up window, ensure Select a file is selected, and then click Select a path to navigate to the location of the downloaded 2ME file.

4. Click Install. The application will then install the workflow. This may take a few minutes as it will install the required containers

5. Successful installation will be indicated with a message. Close the pop-up. The workflow is now installed and ready to use

6. If you wish to test the installation using a small demo dataset provided with the workflow, select wf-hereditary-cancer under the Installed workflows tab. Then, under the Options drop-down menu, select Run demo analysis. Then select Launch.

Running the workflow:

1. Launch the EPI2ME Desktop App.

2. Select wf-hereditary-cancer under the installed workflows tab. Then select Launch.

3. Under Main options, enter the path to the folder containing BAMs for the sample (e.g. barcode01). Please note that the workflow should be run separately for each sample. If you wish to use a name different from the folder name in your final output report, provide the new name in the Sample Name field.

4. Click Launch workflow.

The default values under Multiprocessing Options and Nextflow configuration are suitable for running the workflow on Oxford Nanopore’s PromethION and GridION instruments. We recommend leaving all other parameters at the default values.

wf-hereditary-cancer produces several interactive HTML reports with information on data quality metrics, coverage across target regions, and variants detected. The workflow also outputs additional files that can be used for downstream analysis and exploration, including variant calls (.vcf) for tertiary variant annotation analysis and haplotagged alignment and methylation files (.bam, .bedmethyl) for inspecting the data on a genome browser.

Due to the extended sequencing time, and the multiple flow cell washes and library reloads, we do not recommend re-using the flow cells used in this method.

Re-using these flow cells for subsequent sequencing experiments may result in insufficient data generation for analysis.

Post-run basecalling

If the data was not live basecalled with the recommended HAC model during sequencing, it can be basecalled using the standalone version of the Dorado software. To do so using the command line, please follow the instructions on GitHub page.

You can also basecall your data using our wf-basecalling workflow, available for command line use and via the EPI2ME Desktop App.

Please note that we strongly recommend live basecalling. Post-run basecalling has not been validated at Oxford Nanopore for this Hereditary Cancer Profiling workflow.

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.
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, 250-300 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 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. 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 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.