This protocol aims to rapidly produce libraries with a read N50 of ~30 kb and generate ≥30x coverage of the genome, thereby providing sufficient data to robustly call small and large variants, as well as information on methylation and phasing.

Briefly, genomic DNA is extracted from 500 μl of whole blood samples using the Puregene Blood Kit (Qiagen) and sheared with Megaruptor® 3 (Diagenode). The sheared DNA is then prepared using our Ligation Sequencing Kit V14 (SQK-LSK114) and sequenced across three PromethION™ Flow Cells on a PromethION 24 (P24) or PromethION 48 (P48) device. Sequencing typically runs for 13–16 hours to generate sufficient coverage. At the end of the sequencing, basecalled data is combined into a monolithic BAM file, ready for analysis with the wf-human-variation workflow in EPI2ME. Subsequent VCF files can be imported into either Fabric or Geneyx for a range of tertiary analyses.

Detailed instructions for setting up the sequencing run on MinKNOW™ using the human variation workflow, and importing sequencing data are also included in this protocol.

Steps in the workflow:

The Table below is an overview of the steps required in the workflow, including timings for the processing of samples, and stopping points:

Steps in the workflow	Process	Time	Stop option
Sample preparation	Extract gDNA from blood samples. Fragment the gDNA.	~105 minutes ~45 minutes	At this stage, the extracted gDNA or fragmented gDNA can be stored at –20°C for later use.
Library preparation	DNA repair and end-prep - repair the DNA and prepare the DNA ends for adapter attachment. Adapter ligation and clean-up - attach the sequencing adapters to the DNA ends.	~35 minutes ~20 minutes	4°C overnight We recommend sequencing your library as soon as it is adapted. The DNA library can be stored at 4°C for short-term storage or for repeated use (such as re-loading your flow cell). DNA library can be stored at -80°C for long-term storage.
Sequencing	Prime the flow cell and load the prepared library for sequencing.	~10 minutes per flow cell (3 flow cells per sample)

Prepare for your experiment

You will need to:

• Have the correct consumables, kits, and equipment for DNA extraction, fragmentation, and sequencing. This will include third-party reagents.
• Download the software for acquiring and analysing your data.
• Check your flow cell has sufficient pores for a good sequencing run.

Sample preparation

You will need to:

• Use the recommended protocol to extract genomic DNA (gDNA) from human blood samples, and fragment the gDNA.
• Check the quantity, purity, and length of your extracted material. The quality checks performed during sample preparation are essential in ensuring experimental success.

Library preparation

The Table above outlines the processes involved in library preparation. The quality checks performed during library preparation are essential in ensuring experimental success.

Sequencing and analysis

You will need to:

• Start a sequencing run using the MinKNOW™ software which will collect raw data from the device and convert it into basecalled reads.
• Analyse data using the wf-human-variation workflow from EPI2ME.

Input DNA

For this workflow, we recommend extracting gDNA from 500 μl of whole blood using the Puregene Blood Kit (Qiagen) in the sample preparation step.

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

For the library preparation protocol, you will need ~2.7 µg of fragmented gDNA from each sample.

Third-party reagents

We have validated and recommend the use of all the third-party reagents used in this protocol. Alternatives have not been tested by Oxford Nanopore. For all third-party reagents, we recommend following the manufacturer's instructions to prepare the reagents for use.

Check your flow cell

We highly recommend that you check the number of pores in your flow cells prior to starting a sequencing experiment. This should be carried out within 12 weeks of purchasing your PromethION Flow Cells. Oxford Nanopore will replace any unused flow cell with fewer than the number of pores listed 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 perform 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	5,000

This section describes DNA extraction from 500 µl of whole blood per sample.

Once you have loaded your flow cells, the sequencing run can be started in MinKNOW, the Oxford Nanopore sequencing software that controls the device, data acquisition, and real-time basecalling.

If you do not already have the hg38 reference available on your PromethION device, you can perform the one-time only instructions below in a terminal window, to download the reference and save it to the appropriate directory.

You must generate a BAM file from your sequencing run as this is the required input for the wf-human-variation workflow. Basecalling should be performed using the high-accuracy (HAC) model, with modified basecalling of 5mC and 5hmC in CpG context enabled. The alignment reference should be hg38.

1. Open a terminal window:

   • Ctrl + Alt + T

2. Download the reference:

   • curl -O https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/000/001/405/GCA_000001405.15_GRCh38/seqs_for_alignment_pipelines.ucsc_ids/GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz

3. Uncompress:

   • gunzip GCA_000001405.15_GRCh38_no_alt_analysis_set.fna.gz

4. Move it to the references directory:

   • mv GCA_000001405.15_GRCh38_no_alt_analysis_set.fna ~/data/reference

For trio workflows, set up and run the sequencing separately for each sample (proband + parents), using 3 flow cells per sample. Each sample should be run with its own sample ID and experiment setup.

The steps below demonstrate how to set up your sequencing run in the MinKNOW UI.

1. Navigate to the start page and click Start sequencing.

2. Under the Positions tab, select the positions of the flow cells to be run. Enter an Experiment name and Sample ID.

3. Once the required details have been entered, click Continue at the bottom right of the window.

4. Under the Kit selection tab, select the Ligation Sequencing Kit SQK-LSK114. Then click Continue in the bottom right of the window.

5. In the Run configuration tab, configure the following settings in the Sequencing and analysis panel to enable real-time basecalling, modified base detection, and alignment:

• Enable high-accuracy (HAC) basecalling:

  • Locate the Basecalling section and toggle the switch to ON.

  • From the drop-down menu, select High-accuracy (HAC) as the basecalling model.

  • Confirm your selection by clicking Save.

• Enable modified base detection (5mC/5hmC in CpG).

  • Below the basecalling settings, locate the Modified bases toggle and turn it ON.

  • Select the model for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in CG contexts.

  • Click Save after enabling this option.

• Enable alignment to a reference genome.

  • Scroll to the Alignment section and switch it ON.

  • Click on the white file selection box that appears.

  • Navigate to and select the hg38 reference genome file that you downloaded in the earlier step. This will allow MinKNOW to perform real-time alignment during the run.

  • Click Save once the reference file has been selected.

6. Under the Data targets panel, set the Run limit to 24 hours. Leave the other settings as default and click Continue.

7. In the Output panel, select .BAM format as the output by opening the settings section as indicated below. Also set file output frequency to End of run. Once completed, click Save.

8. Double-check the run parameters and click Start once you are ready to begin your sequencing run. The following section shows some representative sequencing results when using the above parameters.

Figure 1: Read length profile for a 30 kb N50 library. The approximate Gaussian shape is characteristic of genomic DNA that has undergone Megaruptor shearing.

Figure 2: Rate of data accumulation for a library split over three flow cells (3x FCs) with 7,000 (7k) pores (blue), and three flow cells with 5,000 (5k) pores (covered by warranty) (green). Results show that 30x data coverage can be achieved in the quickest time of 12.9 hours of sequencing, using three flow cells with 7,000 pores.

Secondary analysis is performed following sequencing, using the wf-human-variation workflow via EPI2ME or the command line. This variant calling uses:

• Clair3 for single nucleotide variants (SNVs) and small indels

• Sniffles2 for structural variants (SVs)

• Spectre for copy number variants (CNVs)

• Straglr for short tandem repeat expansions (STRs)

• modkit for DNA methylation

The workflow produces per sample VCF files for each variant type (e.g. SNV, SV, STR, CNV). These should be merged as described in the VCF Merging instructions below. Merged VCF files can then be uploaded directly to tertiary interpretation platforms such as Fabric or Geneyx, using your own account credentials with the selected provider.

For trio analysis, this workflow must be run independently for each individual (proband, mother, and father).

1. With the EPI2ME client open on your device, click the Launch button on the side bar.

2. Select the Human Variation workflow.

3. Run Locally and select Launch.

4. In the Workflow Options panel, select all variant types:

• SNP – single nucleotide variants

• SV – structural variants

• CNV – copy number variants

• STR – short tandem repeats

• MOD – modified base summary

If the samples are to be analysed in Fabric, please note that at the time of the production of these instructions, Fabric only supports SNPs and some SVs. However, we recommend choosing all variant types in this analysis for future compatibility purposes in tertiary and as a complete record for the current experiment. A separate VCF file will be generated for each selected workflow.

5. In the Sample name box, provide the Sample ID which you used in MinKNOW. Remember that we specified an identical sample ID for all three flow cells which were loaded with the same sample. This should be replicated here, for the workflow to find and merge all sets of data related to this sample.

6. Select the folder under Input: BAM file. This opens a file browser window. Find and select your experiment folder. Then click Open to complete.

7. Select the Reference file. Navigate using the file browser window and select the reference file you downloaded earlier. Then click Open.

8. Select the Phasing checkbox if you want phased variant calls. In the Partner integration drop-down menu, choose either Geneyx or Fabric. This step ensures the VCF output is compatible for upload with the chosen tertiary analysis provider. For Geneyx analysis, two VCF files will be written out (SNPs and all other variant types – CNV, SV, STR), whereas for Fabric, a single VCF file merging all variants will be made available.

9. Once you have provided the appropriate sample name, a path to the experiment folder, a reference file, and selected your tertiary partner, the Main options selection will show a green tick. Double-check everything and then click Launch workflow in the bottom right.

10. To check that your analysis is running properly, go to the Results section, find your analysis and click Details. After a delay of ~20 seconds, progress bars will appear showing the active processes.

11. Once the analysis is complete, navigate to the Files tab within the run dashboard. All outputs, including reports, VCFs, and intermediate files are listed here.

• You can Open files directly (e.g. VCFs, reports) to review results within the interface.

• Alternatively, select Open in explorer to access the files in their local directory for downstream analysis or transfer.

• The files created for uploading to Fabric/Geneyx (if chosen) will be in a subdirectory inside the output folder called integrations/TertiaryPartner where the latter is either Fabric or Geneyx.

Once the wf-human-variation analysis has completed successfully, the resulting VCF should be uploaded to either Fabric or Geneyx for tertiary analysis.

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.

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.