Download the MinKNOW Software for the MinION Mk1B/D and the PromethION 2 Solo (P2 Solo) Devices from the Software Downloads page and extract the .zip files. Follow the installation prompts to install the software.

The MinKNOW App is also available to download for iOS and Android devices to enable you to monitor experiment progress remotely.

Connect your device to the host computer via the USB cable provided, as instructed in the user manual. Please refer to MinION Mk1B Computer requirements or MinION Mk1D Computer requirements for further information.

1. Double-click on the MinKNOW icon on your desktop.

2. Click on Log in with your Nanopore account.

3. Enter your email address and Nanopore password.

4. The Connection Manager will open, and your device will be the card in the top left corner.

5. Click on the My device card to access and control it.

A hardware check must be completed on your new device or after software has been updated.

1. Open the MinION Mk1B/Mk1D and slide your Configuration Test Cell (CTC) under the clip. Gently press down onto the CTC until it clicks into place. To check a Flongle Adapter, insert an empty adapter instead of a CTC.

2. Close the device lid.

3. Navigate to the Start tab and click Hardware check.

4. Click Start. The hardware check will complete after approximately 1 minute.

5. After the hardware check has finished, remove the CTC and store in dry conditions.

A flow cell check must be carried out prior to starting a sequencing experiment to assess the number of active pores available in your flow cell. For further information about flow cell warranty is available in the Flow cell check section.

1. Open the device and slide your flow cell under the clip. Firmly press down on the flow cell. For Flongle, slide the Flongle Adapter under the clip and insert a Flongle Flow Cell.

2. Navigate to the Start page and click Flow Cell Check.

3. MinKNOW will recognise the MinION Flow Cell type and IDs. Note: For Flongle, fill in the flow cell ID manually.

4. Click Start and the flow cell check will begin.

Prime and load your flow cell with DNA or RNA library by following a library prep protocol and set up a sequencing run, as follows:

1. Navigate to the Start page and click Start Sequencing.

2. In the Positions tab, fill in the experiment name, sample ID, and select the flow cell type. Click Continue to kit selection. Note: For Flongle, fill in the flow cell ID manually.

3. Select the kit and any expansion(s) used to prepare the library. Click Continue to run options.

4. Specify the sequencing run length and minimum read length or keep the default settings. Click Continue to analysis.

5. Choose your basecaller model and select any available barcoding and alignment options or keep the default settings. Click Continue to output.

6. Specify your output data location, format and filtering options or keep the default settings. Click Continue to final review.

7. Click Start.

For further details, please see the Starting a sequencing run section for your device in the MinKNOW protocol.

1. Connect the AC/DC adapter to a power source and connect the MinION Mk1C.

2. Switch the power source on and press the power button on the device.

3. For the first time, users will be taken through the onboarding process to set up the device. Through this process, you will:

   • Connect your device to the internet (Ethernet recommended)
   • Log into your MinKNOW account using your Community credentials
   • Configure local network access to your device
   • Perform a hardware check

Verify the time and data of your device is correct to successfully establish a network connection.

1. Navigate to Device settings in the host settings.

2. Check the time and date.

3. Click Change data and time and set the time using custom date/time.

The device is shipped with a default password minit that must be reset.

1. Navigate to Device settings in the host settings.

2. Click Reset password to open the Change password dialogue box.

3. Enter the current password, followed by the new password.

4. Click Reset password to confirm the password change.

A hardware check must be performed on new devices or when software has been updated.

1. Open the MinION Mk1C and slide the CTC under the clip. Gently press down onto the CTC until it clicks into place. To check a Flongle Adapter, insert an empty adapter instead of a CTC.

2. Close the device lid.

3. Navigate to the Start page and click Hardware check.

4. Click Start. The hardware check will complete after approximately 1 minute.

5. After the hardware check has finished, remove the CTC and store in dry conditions.

A flow cell check must be carried out prior to starting a sequencing experiment to assess the number of active pores available in your flow cell. For further information about flow cell warranty is available in the Flow cell check section.

1. Open the device and slide your flow cell under the clip. Firmly press down on the flow cell. For Flongle, slide the Flongle Adapter under the clip and insert a Flongle Flow Cell.

2. Navigate to the Start page and click Flow Cell Check.

3. MinKNOW will recognise the MinION Flow Cell type and IDs. Note: For Flongle, fill in the flow cell ID manually.

4. Click Start and the flow cell check will begin.

Prime and load your flow cell with DNA or RNA library by following a library prep protocol and set up a sequencing run, as follows:

1. Navigate to the Start page and click Start Sequencing.

2. In the Positions tab, fill in the experiment name, sample ID, and select the flow cell type. Click Continue to kit selection. Note: For Flongle, fill in the flow cell ID manually.

3. Select the kit and any expansion(s) used to prepare the library. Click Continue to run options.

4. Specify the sequencing run length and minimum read length or keep the default settings. Click Continue to analysis.

5. Choose your basecaller model and select any available barcoding and alignment options or keep the default settings. Click Continue to output.

6. Specify your output data location, format and filtering options or keep the default settings. Click Continue to final review.

7. Click Start.

For further details, please see the Starting a sequencing run section for your device in the MinKNOW protocol.

Unpack the device in an appropriate laboratory location and attach all the required cables to the device, including the:

• Ethernet
• HDMI or DisplayPort to monitor
• USB for a keyboard
• USB for a mouse
• C13 cable

Ensure the device is connected to a protective earth mains supply and turn the power source on and press the power button on the device. A blue light will appear in the middle of the button.

1. Double-click on the MinKNOW icon on your desktop.

2. Click on Log in with your Nanopore account.

3. Enter your email address and Nanopore password.

4. The Connection Manager will open, and your device will be the card in the top left corner.

5. Click on the My device card to access and control it.

To connect your GridION to a single network, use the user interface. A device can be connected to multiple networks which will require a script to be used. For more information, please see the GridION User Manual.

Validate your IP address to ensure it has been correctly set using a static or DHCP address using the command ifconfig.

Verify the time and data of your device is correct to successfully establish a network connection.

1. Navigate to Device settings in the host settings.

2. Check the time and date.

3. Click Change data and time and set the time using custom date/time.

The device is shipped with a default password grid that must be reset.

1. Navigate to Device settings in the host settings.

2. Click Reset password to open the Change password dialogue box.

3. Enter the current password, followed by the new password.

4. Click Reset password to confirm the password change.

A hardware check must be perfomed on new devices or after software has been updated.

1. Open the GridION and slide a CTC under the clip for each position. Gently press down onto the CTC until it clicks into place. To check a Flongle Adapter, insert an empty adapter instead of a CTC.

2. The LEDs above the flow cell position will be blue when initialising and will turn green when they are ready.

3. Close the device lid.

4. Navigate to the Start page and click Hardware check.

5. Click Select all available positions.

6. Click Start. The hardware check will complete after approximately 1 minute.

7. After the hardware check has finished, remove the CTCs and store in dry conditions.

A flow cell check must be carried out prior to starting a sequencing experiment to assess the number of active pores available in your flow cell. For further information about flow cell warranty is available in the Flow cell check section.

1. Open the device and slide your flow cell under the clip. Firmly press down on the flow cell. For Flongle, slide the Flongle Adapter under the clip and insert a Flongle Flow Cell.

2. Navigate to the Start page and click Flow Cell Check.

3. MinKNOW will recognise the MinION Flow Cell type and IDs. Note: For Flongle, fill in the flow cell ID manually.

4. Click Start and the flow cell check will begin.

Prime and load your flow cell with DNA or RNA library by following a library prep protocol and set up a sequencing run, as follows:

1. Navigate to the Start page and click Start Sequencing.

2. In the Positions tab, fill in the experiment name, sample ID, and select the flow cell type. Click Continue to kit selection. Note: For Flongle, fill in the flow cell ID manually.

3. Select the kit and any expansion(s) used to prepare the library. Click Continue to run options.

4. Specify the sequencing run length and minimum read length or keep the default settings. Click Continue to analysis.

5. Choose your basecaller model and select any available barcoding and alignment options or keep the default settings. Click Continue to output.

6. Specify your output data location, format and filtering options or keep the default settings. Click Continue to final review.

7. Click Start.

For further details, please see the Starting a sequencing run section for your device in the MinKNOW protocol.

1. Unpack the device and place the Sequencing Unit and the Data Acquisition Unit within 2 meters of each other.

2. Connect the following cables:

• Connect either the copper or fibre cables to the data acquisition unit.
• Connect the PCIe and USB cables between the sequencing and data acquisition units
• Connect the peripheral devices (e.g. monitor, mouse, keyboard) to the appropriate ports.
• Connect the two C13 cables to both units.

3. Turn on the power at the mains supply and turn on the sequencing unit, wait three minutes and turn on the data acquisition unit.

4. Validate your IP address to ensure it has been correctly set using a static or DHCP address using the command ifconfig.

1. Double-click on the MinKNOW icon on your desktop.

2. Click on Log in with your Nanopore account.

3. Enter your email address and Nanopore password.

4. The Connection Manager will open, and your device will be the card in the top left corner.

5. Click on the My device card to access and control it.

Verify the time and data of your device is correct to successfully establish a network connection.

1. Navigate to Device settings in the host settings.

2. Check the time and date.

3. Click Change data and time and set the time using custom date/time.

The device is shipped with a default password prom that must be reset.

1. Navigate to Device settings in the host settings.

2. Click Reset password to open the Change password dialogue box.

3. Enter the current password, followed by the new password.

4. Click Reset password to confirm the password change.

A hardware check must be performed on new devices or after a software update.

1. Open the PromethION and insert the CTCs into all positions. The LED adjacent to the flow cell will be blue when initialising and will turn green when it is ready.

2. Close the device lid.

3. Navigate to the Start page and click Hardware check.

4. Click Select all available positions.

5. Click Start. The hardware check will complete after approximately 1 minute.

6. After the hardware check has finished, remove the CTCs and store in dry conditions.

A flow cell check must be carried out prior to starting a sequencing experiment to assess the number of active pores available in your flow cell. For further information about flow cell warranty is available in the Flow cell check section.

1. Open the device and insert your flow cell.

2. Navigate to the Start page and click Flow Cell Check.

3. MinKNOW will recognise the PromethION Flow Cell type and ID.

4. Click Start and the flow cell check will begin.

Prime and load your flow cell with DNA or RNA library by following a library prep protocol and set-up a sequencing run, as follows:

1. Navigate to the Start page and click Start Sequencing.

2. In the Positions tab, fill in the experiment name, sample ID, and select the flow cell type and position(s). Click Continue to kit selection.

3. Select the kit and any expansion(s) used to prepare the library. Click Continue to run options.

4. Specify the sequencing run length and minimum read length or keep the default settings. Click Continue to analysis.

5. Choose your basecaller model and select any available barcoding and alignment options or keep the default settings. Click Continue to output.

6. Specify your output data location, format and filtering options or keep the default settings. Click Continue to final review.

7. Click Start.

For further details, please see the Starting a sequencing run section for your device in the MinKNOW protocol.

1. Place the P2 Solo and GridION on a laboratory bench.

2. Check the GridION serial number is greater than GXB002xxx on the back of the device.

3. Download and install the PromethION 2 Solo software from the Software Downloads page on the Community. For Linux, install the software via the Terminal. For more information, see the PromethION 2 Solo User Manual. Note: The MinKNOW App is also available to download for iOS and Android devices to allow you to monitor experiment progress and to control your device remotely.

4. Connect the P2 Solo AC power adapter into a mains supply and connect it to the P2 Solo.

5. Connect the USB Type-C cable to the GridION and the P2 Solo.

6. Turn on the power switch on the GridION and wait 60 seconds.

1. Place the P2 Solo on a laboratory bench.

2. Download and install the PromethION 2 Solo software from the Software Downloads page on the Community. For Linux, install the software via the Terminal. For more information, see the PromethION 2 Solo User Manual. Note: The MinKNOW App is also available to download for iOS and Android devices to allow you to monitor experiment progress and to control your device remotely.

3. Connect the P2 Solo AC power adapter into a mains supply and connect it to the P2 Solo.

4. Connect the USB Type-C cable to the workstation/laptop and the P2 Solo.

1. Double-click on the MinKNOW icon on your desktop.

2. Click on Log in with your Nanopore account.

3. Enter your email address and Nanopore password.

4. The Connection Manager will open, and your device will be the card in the top left corner.

5. Click on the My device card to access and control it.

For your P2 Solo connected to a GridION, the network will need to be configured. To connect your GridION to a single network, use the user interface. A device can be connected to multiple networks which will require a script to be used.

Validate your IP address to ensure it has been correctly set using a static or DHCP address using the command ifconfig.

A hardware check must be performed on new devices and after software updates.

1. Open the P2 Solo and slide the CTC into each position. Note: Ensure you are using the CTCs provided with the P2 Solo device only.

2. Close the device lid.

3. Navigate to the Start page and click Hardware check.

4. Click Select all available positions.

5. Click Start. The hardware check will complete after approximately 1 minute.

6. After the hardware check has completed, remove the CTCs and store in dry conditions.

A flow cell check must be carried out prior to starting a sequencing experiment to assess the number of active pores available in your flow cell. For further information about flow cell warranty is available in the Flow cell check section.

1. Open the device and insert your flow cell.

2. Navigate to the Start page and click Flow Cell Check.

3. MinKNOW will recognise the PromethION Flow Cell type and ID.

4. Click Start and the flow cell check will begin.

Prime and load your flow cell with DNA or RNA library by following a library prep protocol and set-up a sequencing run, as follows:

1. Navigate to the Start page and click Start Sequencing.

2. In the Positions tab, fill in the experiment name, sample ID, and select the flow cell type and position(s). Click Continue to kit selection.

3. Select the kit and any expansion(s) used to prepare the library. Click Continue to run options.

4. Specify the sequencing run length and minimum read length or keep the default settings. Click Continue to analysis.

5. Choose your basecaller model and select any available barcoding and alignment options or keep the default settings. Click Continue to output.

6. Specify your output data location, format and filtering options or keep the default settings. Click Continue to final review.

7. Click Start.

For further details, please see the Starting a sequencing run section for your device in the MinKNOW protocol.

1. Download MinKNOW software from the Downloads page on the Community
2. Follow the on-screen instructions
3. Contact the local IT administrators if there are any issues with permissions for installation

1. Check that the Oxford Nanopore wheel icon is next to MinKNOW Stand-alone NC Windows [version number].zip
2. Click on MinKNOW Stand-alone NC Windows [version number].zip

In the Choose Data Location dialogue box, check that the read location is C:\data. If you prefer a different destination, enter the path in the box. Continue the download by clicking on Install.

1. Download MinKNOW software from the Downloads page on the Community. 2. Once MinKNOW has downloaded, navigate to the folder in Applications and launch the MinKNOW.pkg installer.

3. Follow the on-screen instructions.

4. Provide a fingerprint or password as required.

5. Once the installation is complete, exit the installer.

For Ubuntu 22:

sudo apt update
sudo apt install wget
wget -O- https://cdn.oxfordnanoportal.com/apt/ont-repo.pub | sudo apt-key add -
echo "deb http://cdn.oxfordnanoportal.com/apt jammy-stable non-free" | sudo tee /etc/apt/sources.list.d/nanoporetech.sources.list 

For Ubuntu 20:

sudo apt update
sudo apt install wget
wget -O- https://cdn.oxfordnanoportal.com/apt/ont-repo.pub | sudo apt-key add -
echo "deb http://cdn.oxfordnanoportal.com/apt focal-stable non-free" | sudo tee /etc/apt/sources.list.d/nanoporetech.sources.list

sudo apt update
sudo apt install ont-standalone-minknow-release

Select Get Update to update the device software automatically.

Updates may be skipped. However, we recommend to update the device as soon as updates are available. Some updates will be mandatory to use the device and are unable to be skipped.

Note: For MinION Mk1B/Mk1D, clicking Get Update will open the Software Downloads page on the connected computer for you to download the updated MinION software.

sudo apt clean
sudo apt update 
sudo apt install ont-standalone-minknow-release

The latest version of MinKNOW will be installed. Once these commands have completed successfully without errors, reboot the device:

sudo reboot

To log in, you must be connected to the internet.

From here, you can also change the language in the user interface in the bottom right corner.

You will be prompted to enter your email address and Nanopore password:

If you experience login issues, please visit the Community Support channel (https://community.nanoporetech.com/support) and use Continue as guest for temporary use.

• Click the button in the top right corner, labelled with your initials or 'Guest' and click Logout.

This example shows a Flongle Flow Cell inserted in a MinION Mk1C device.

Select options for local and remote access.

This example is on the MinION device with a Flongle adapter. Note: To check the Flongle adapter, insert the EMPTY adapter. To check device or position, insert just a MinION CTC - the Flongle adapter is not needed.

This example is on the GridION device. Ensure to select the postition(s) with a CTC inserted before starting hardware check.

This example is on a GridION device:

Note: For Flongle, the flow cell ID must be filled in manually.

To transfer a configuration file between devices, it can be exported to a JSON file by clicking Export, or imported by clicking Import.

Note: Reboot is unavailable on GridION but the software can be restarted.

Data will now be able to be shared and accessible on different networks.

Note: The image above is an example of sharing on a GridION. On MinION devices, 'minit' will appear and PromethION devices will show 'prom' when active.

Note: a USB drive or SD card plugged in will appear under the Devices folder.

Use SMB for connecting to a shared drive on a Windows server, and NFS for Linux. If your shared drive is on a macOS machine or NAS (Network-Attached Storage), you can use either SMB or NFS.

SMB:

NFS:

1. Enter the host name or IP address of the SMB share. This is acquired from your network admin.
2. Enter the drive on the network host that the user wants to share. Take note to start with a '/'. E.g. /data.
3. The 'Mount Point' field will auto-populate with the host name and path to the network share. However, you can customise this. In the file manager, it will appear as: /data/network/mt-111111-data for example.
4. Add a username.
5. (Optional) Enter a password.
6. (Optional) Enter a domain name.
7. Click Mount.

Providing the settings were correct, the mounted drive will be added to the 'Disk Management' overview. The user will be able to:

• View the space information for the drive
• Unmount the network drive
• A link to jump to the mount point inside the file manager

1. Enter the host name or IP address of the NFS share. Obtain this from your network admin.
2. Enter the drive on the network host that the user wants to share. Take note to start with a '/'. E.g. /data.
3. The 'Mount Point' field will auto-populate with the host name and path to the network share. However, you can customise this. In the file manager, it will appear as: /data/network/mt-111111-data for example.
4. Click Mount.

Providing the settings were correct, the mounted drive will be added to the 'Disk Management' overview. You will be able to:

• View the space information for the drive
• Unmount the network drive
• A link to jump to the mount point inside the file manager

Ensure the experiment name and sample ID do not contain any personally identifiable information.

Note: If sample ID is not filled in, there will be no sample ID in the folder name structure.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Select Continue to move to the Kit Selection page.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you will see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs can be generated manually by clicking Export run report and selecting which experiment to export to html.

Run reports are automatically generated for Flongle running on MinION Mk1C and MinION Mk1B/Mk1D running on Linux. However, for MinION Mk1B running on Windows or Mac, the run reports need to be manually generated.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B/Mk1D.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Select Continue to move to the Kit Selection page.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you will see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs can be generated manually by clicking Export run report and selecting which experiment to export to html.

Run reports are automatically generated for MinION Mk1B/Mk1D running on Linux. However, for MinION Mk1B running on Windows or Mac, the run reports need to be manually generated.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B/Mk1D.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Select Continue to move to the Kit Selection page.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you can see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs are automatically generated but can be generated manually by clicking Export run report and selecting which experiment to export to html.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B/Mk1D.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

Select Continue to move to the Kit Selection page.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you can see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs are automatically generated. To manually generate a run report, click Export run report and selecting which experiment to export to html.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B.

Select Continue to move to the Kit Selection page.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Use "Select all available" to select all the connected flow cells or use the diagram above to select specific flow cells for an experiment.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

By default, the analysis workflow option is turned off.

To enable this feature, switch the Workflow toggle on. Select a workflow to add to your experiment and click 'Select workflow' to continue.

From the Review workflow settings window, you can select from the Workflow options and import Human genome reference files. To use the Methylation sub-workflow, Modified base calls must be enabled.

Input options are limited in comparison to the EPI2ME desktop application. The desktop version should be used to access advanced options. It is recommended that only one workflow is run at a time. The workflow outputs a VCF file which will be saved to the experiment directory. When the workflow is complete, a link to the workflow report will appear in the MinKNOW experiments page.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you can see a progress bar below each flow cell to show progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first channel scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs are automatically generated but can be generated manually by clicking Export run report and selecting which experiment to export to html.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B.

Select Continue to move to the Kit Selection page.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Use "Select all available" to select all the connected flow cells or use the diagram above to select specific flow cells for an experiment.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you can see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs are automatically generated. To manually generate a run report, click Export run report and selecting which experiment to export to html.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B.

Select Continue to move to the Kit Selection page.

Ensure the experiment name and sample ID does not contain any personally identifiable information.

To use previously saved settings, please see the "Save sequencing run settings" page of this protocol.

Use "Select all available" to select all the connected flow cells or use the diagram above to select specific flow cells for an experiment.

Note: If sample ID is not filled in, there will be no sample ID in the folder structure.

One sequencing kit can be selected as well as one or more expansions. If a sequencing kit does not have any associated expansions, no expansion options will appear.

The filter options may be used to find the kit used. For example, click DNA to filter for DNA sequencing kits.

For control runs, there is a Control checkbox which only filters for the control experiments of the kits. This will automatically set the sequencing parameters for a control run. Note: Run length will be reduced to 6 hours for control experiments.

Click Continue to navigate to the run configuration page.

At the top of the run configuration page you will see an overview of your experiment set-up and identifiers inlcuding:

• Experiment name: defined by user.
• Positions: Flow cell position(s) being used in the sequencing experiment, and the associated sample IDs for the position(s).
  Note: If using multiple flow cells, click "show details" to open a window displaying all the positions used and the associated sample IDs.
• Flow cell type: automatically detected by the MinKNOW software.
• Sequencing kit: the sequencing kit used for DNA/RNA library preparation, user-defined.

The basecalling option is on by default to perform real-time basecalling. This can be switched off and basecalling performed post-sequencing.

Note: If basecalling is disabled, barcoding and alignment will not be performed during sequencing.

Modified bases is off by default. This can be switched on to use the CpG context models and basecall 5mC and 5hmC.

To specify your basecall model and modified bases, click the Edit icon to open basecalling options dialogue box.

Barcoding options are only available when a barcoding sequencing kit or expansion has been selected. Barcoding can be switched off and performed post-sequencing.

To specify the barcoding options, click the Edit icon to open the barcoding options dialogue box. From here, barcode trimming and custom barcode selection can be defined.

By default, barcoding trimming is off. This can be enabled to remove barcodes from demultiplexed reads. Please note, some primer sequences may also be trimmed together with the barcodes.

By default, custom barcode selection is off. This can be enabled to only select for specified barcodes used in your library preparation for the current sequencing run.

Note: Specifying barcodes must be entered as:

• Individual numbers.
• Numbers separated by commas.
• Range of numbers separated by a dash.
• A combination of numbers and ranges, separated by commas.

Some sequencing kits place barcodes on both ends of the read. By default, the barcode both ends feature is off. If this feature is enabled, the barcode will only classified if a barcode above the minimum barcode score is present at both ends of the basecalled read.

Note: For barcoding to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

To use alignment during sequencing, upload an alignment reference file as a .fasta or .mmi file. We recommend uploading a reference locally on bacterial-sized genomes. Uploading a file can take a few minutes and is compute-dependent.

A reference file can contain multiple entries in the same file (e.g. multiple chromosomes) and alignment hits from these files are used to populate the alignment graphs which can be viewed on the GUI.

A BED file can also be uploaded alongside the reference when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome). Alignment hits from BED files will be highlighted in the sequencing .txt file generated in the data folder. Click Edit options to open a dialogue box to upload a BED file.

BED files will be validated upon upload. If there is an error in the validation of the file, an error message will show linking to a web page with guidance on correcting the validation errors.

Note: For alignment to be performed during sequencing, basecalling must be enabled.

By default, alignment is turned off.

Enabling adaptive sampling will allow you to enrich or deplete specific sequences from your aligned sequencing data.
For our barcoding kits, the barcode balancing feature is also available using adaptive sampling through the "beta" features in MinKNOW.

By default, all adaptive sampling reads, whether they are accepted or rejected, will be basecalled. If you would like to only basecall on-target reads and not rejected reads, set the Basecally on-target reads only toggle to On. If you enable raw data generation, rejected reads will still be written out but not basecalled.

More information on this feature can be found in our Adaptive sampling documentation.

This feature allows you to set up the rules to define when a sequencing run will stop. If multiple rules are set up, the first rule met will stop the run.

Click the Edit icon to open the options.

Run limit

This rule must be defined based on run time length in hours or when a flow cell is end of life and has no more sequencing pores available.

Use the drop-down menu to choose:

• "Time equals" and hours
• or "Flow cell is" which will automatically be set as "End of life".

Flow cell data target

This rule can be used to define a target amount of data to reach before a sequencing run is stopped. This can be specified for either estimated or basecalled bases and your data target will be available to view on the experiment graphs to allow you to monitor data progress.

Please note, basecalling must keep up with data output in real-time. If the device or basecall model will not keep up and reach the target output before the run limit rule is met, we recommend using the "Estimated bases" option. However, if your experiment will not reach the target data before the end of the experiment as defined by the run limit rule, you will be warned by the UI.

Use drop-down menu to select for "Estimated bases equal" or "Basecalled bases equal" and input the value in either Gb or Mb.

The output section provides variables for data output including file name, location and format.

Click the Edit icon to open the options.

Output settings:

Data saved as:

Check the name of the output file for the data.

Output location:

Confirm the location for the output data file. Click the file path name to open a pop-up to alter file path or create a new file.

Click Choose location on the pop-up to confirm new file location.

Output location storage capacity:
A bar indicator is displayed below the selected data output file location. This bar will display red when the disk space used reaches more than 80% of the total available disk space. Otherwise, the bar will display green. Below the bar, you can see how much drive space is available.

Basecalled output options:

Confirm output file type and file compression using the checkboxes.

Note: Below, reads per file can be changed for both raw reads and basecalled reads, but take note:

• Fewer reads per file: reads become more quickly available. However, too few reads per file means MinKNOW may not keep up with writing out files in real-time.
• More reads per file: reduces the number of files especially for amplicon/cDNA experiments that produce a large number of reads. Some downstream analysis tools may have an upper limit on the uploaded file size.

Basecalled output type:
Output file type can be selected between .FASTQ or .BAM files. The .FASTQ will be on as default if basecalling in real-time.

File output frequency can be adjusted for basecalled files based on two options:

For barcoding runs, basecalled reads are split into files by barcode by default.

Gzip compression of FASTQ files is on by default of basecalled reads. Note: FASTQ Gzip files may prevent some downstream informatics applications.

Raw reads output options:

Raw reads: Raw reads output can be saved as .POD5 or .FAST5.

• For Kit 14 chemistry and SQK-RNA004, .POD5 is the default file output. The .POD5 output generates files hourly.
• The .FAST5 output file is the default format for our previous and "legacy" chemistry kits. The.FAST5 output produces files for every 4000 reads.

.POD5 is a Nanopore-developed file format which stores Nanopore data in a more accessible way and can be used as an alternative to FAST5 output. This output also writes data faster, uses less compute and has smaller raw data file size.

We recommend keeping the default settings.

Raw reads split by barcode: Raw read files generated in the .POD5 format can now be split by barcode (if using one of our barcoding sequencing kits.
Please note, this feature is turned off by default and not available for .FAST5 files.

Filtering options can be used to determine which reads are classed into pass or fail files. These options may also be used to determine which predefined reads, read lengths and Qscore during basecalling can be split out in some live graphs.

Click the Edit icon to open the filtering options:

Use the GUI to alter Qscore and to define minimum and maximum readlength for a pass read, if required.

From here, you can see a progress bar below the flow cell to show the progression of the sequencing script.

In some cases, the device will take a few minutes before starting a sequencing run if an alignment reference file is used. A progress bar will show the progress before sequencing begins.

Flow cell health will be displayed after the first pore scan.

Below are recommended troubleshooting tips if there are unexpected differences in pore numbers:

• If there is a significant reduction in active pores in the first pore scan, restart MinKNOW.
• If the numbers are still significantly different, close down the host computer and reboot.
• When the numbers are similar to those reported at the end of the flow cell check, restart the experiment. There is no need to load any additional library after the restart.

This is only necessary if you want to stop your run before the end of the set run time.

Confirm to stop experiment by clicking:

• Stop sequencing which will start catch-up basecalling
• or Stop sequencing and basecalling which will stop both sequencing and basecalling immediately.

A run report containing information about the sequencing run and performance graphs are automatically generated. To manually generate a run report, click Export run report and selecting which experiment to export to html.

For more information about the run report, please see the Run report section of this document.

A pore activity .csv file is also generated for every run.

The report and .csv files are saved to the same folder as the .fast5 and .fastq read files e.g. :\data\experiment\sample_ID\ for MinION Mk1B.

An indication no experiments are running is the lack of an experiment status bar beneath individual flow cells.

An indication no experiments are running is the lack of an experiment status bar beneath individual flow cells.

Select whether you want to process reads in sub-directories.

If your chosen read input folder contains sub-directories with FAST5 files, you can choose whether to basecall files in these sub-directories by enabling this option.

By default, MinKNOW will create a /basecalled folder in /data. You can set a different folder in which to save the basecalled reads. Note: This must be a sub-folder of /data directory.

By default, output FASTQ files will be compressed using Gzip. This feature can be toggled off by the user.

Set-up the configuration of your basecalling run using the drop-down menus:

• In the run setting, select the parameters used to initially sequence the data.
• In the basecalling settings, select the basecall model used to process raw signal, and whether to use modified basecalling.

Once all the parameters have been selected, the basecalling configuration will be displayed in the lower panel.

Given the recent improvements to basecalling accuracy, Q-score cut-off for reads put into the "pass" folder has been increased to:

Fast basecalling model >8 High accuracy (HAC) model >9 Super accurate (SUP) model >10

Select whether you want to process FASTQ reads in sub-directories.

If your chosen read input folder contains sub-directories with FASTQ files, you can choose whether to barcode FASTQ files in these sub-directories by switching this option on.

Note: Data can only be saved to the /data folder.

By default, MinKNOW will create a /barcoding folder in /data/. You can set a different folder which to save the barcoded reads. Note: This must be a sub-folder of /data directory.

You can also select your output file formats to be generated as .BAM or .FASTQ.

MinKNOW will write out basecalled reads into barcode-specific folders and enable demultiplexing during basecalling.

Users are able to choose specific settings, including trimming barcodes and barcoding both ends (available for specific barcoding kits).

Select whether you want to process FASTQ reads in sub-directories.

If your chosen read input folder contains sub-directories with FASTQ files, you can choose whether or not to align FASTQ files in these sub-directories by switching this option on.

Note: Data can only be saved to the /data folder.

By default, MinKNOW will create an /alignment/ folder in /data/. You can set a different folder which to save the aligned reads. Note: This must be a sub-folder of /data directory.

The alignment reference must either be a FASTA file.

However, there is also the option to use a BED file alignment reference as well.