MinKNOW

Overview

Instructions for working with MinKNOW. If using for the first time, please refer to the user manual for your device. For Research Use Only

Document version: MKE_1013_v1_revDF_22Nov2024

1. Quick start guides introduction

Introduction

MinKNOW controls all Oxford Nanopore sequencing devices, performing several core tasks, including data acquisition, real-time analysis, basecalling, and data streaming.

In this section, we will outline how to set up your device for the first time for sequencing using our MinKNOW software.

2. MinION Mk1B and MinION Mk1D quick start guides

The MinION Mk1B and MinION Mk1D

The MinION Mk1B and MinION Mk1D are pocket-sized, portable devices for biological analysis of DNA or RNA samples. The MinION Mk1D is the successor to MInION Mk1B, with improved thermal dissipation capabilities significantly enhancing sequencing performance, enabling highly accurate real-time sequencing in an even wider range of environments than MinION Mk1B. Both devices can be used with MinION Flow Cells, as well as the Flongle Adapter and flow cells. The device is plugged into a computer and is operated by MinKNOW, which controls the device and flow cells, collects the sequencing data, and performs basecalling.

Below is a quick start guide to set up a new device for sequencing library samples. For further details on using these devices, please refer to the MinION Mk1B user manual or MinION Mk1D user manual.

Download and install the MinKNOW software.

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.

Set up your MinION Mk1B/Mk1D with a host computer.

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.

Log into MinKNOW.

  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.

Min login

Tutorials

These are available to help you get started and run your experiments as easily as possible.

When you open the MinKNOW software for the first time, the tutorials will navigate you through the graphical user interface (GUI). Click Continue tutorial to follow or click the to skip.

tutorial

Perform a hardware check.

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.

Hardware check

Perform a flow cell check.

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.

Min flow cell check

Set up a sequencing run.

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.

start

3. MinION Mk1C quick start guide

The MinION Mk1C

This device combines the real-time, rapid, portable sequencing of a MinION Mk1B/Mk1D with a Graphical Processing Unit (GPU) and a high-resolution screen. It can be used with MinION Flow Cells, as well as the Flongle Adapter and flow cells. The device is operated by the MinKNOW software that controls the device, experimental scripts, and performs basecalling.

Below is a quick start guide to set up a new device and to start sequencing your library samples. For further details using this device, please see the MinION Mk1C User Manual.

Set up the MinION Mk1C device.

  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

Check the time and date of your device.

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.

Check time

Reset the default password.

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.

reset password

Tutorials

These are available to help you get started and run your experiments as easily as possible.

When you open the MinKNOW software for the first time, the tutorials will navigate you through the graphical user interface (GUI). Click Continue tutorial to follow or click the to skip.

tutorial

Perform a hardware check.

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.

Hardware check

Perform a flow cell check.

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.

Min flow cell check

Set up a sequencing run.

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.

start

4. GridION quick start guide

The GridION

This is a compact benchtop system offering on-demand sequencing with integrated real-time data processing. Up to five MinION or Flongle Flow Cells can be run individually or concurrently, offering a cost-effective access to long-read, real-time nanopore sequencing. The device is operated by the MinKNOW software, which controls the device, experimental scripts, and performs basecalling.

Below is a quick start guide to set up a new device and to start sequencing your library samples. For further details using this device, please see the GridION User Manual.

Install the GridION device.

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.

Log into MinKNOW.

  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.

Min login

Tutorials

These are available to help you get started and run your experiments as easily as possible.

When you open the MinKNOW software for the first time, the tutorials will navigate you through the graphical user interface (GUI). Click Continue tutorial to follow or click the to skip.

tutorial

Configure your GridION.

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.

Check the time and date of your device.

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.

Check time

Reset the default password.

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.

reset password

Perform a hardware check.

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.

Grid hardware check

Perform a flow cell check.

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.

Grid flow cell check

Set up a sequencing run.

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.

start

5. PromethION 24/48 quick start guide

The PromethION 24 and 48

This is a benchtop device designed to run up to 24 (for P24) or 48 (for P48) flow cells. Each flow cell is independently addressable, meaning that experiments can be run concurrently or individually. The integrated compute in the PromethION enables real-time data streaming and analysis. The device is operated by the MinKNOW software that controls the device, experimental scripts, and performs basecalling.

Below is a quick start guide to set up a new device and to start sequencing your library samples. For further details using this device, please see the PromethION 24/48 User Manual.

Install the PromethION device.

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

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

Log into MinKNOW.

  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.

Min login

Tutorials

These are available to help you get started and run your experiments as easily as possible.

When you open the MinKNOW software for the first time, the tutorials will navigate you through the graphical user interface (GUI). Click Continue tutorial to follow or click the to skip.

tutorial

Check the time and date of your device.

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.

Check time

Reset the default password.

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.

reset password

Perform a hardware check.

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.

Prom hardware check

Perform a flow cell check.

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.

Prom flow cell check

Set up a sequencing run.

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.

start

6. PromethION 2 Solo quick start guide

The PromethION 2 Solo

This is a small benchtop device designed to run up to two flow cells. Each flow cell is independently addressable, meaning that experiments can be run concurrently or individually. The PromethION 2 Solo plugs into a GridION Mk1 or user compute for real-time data streaming and analysis. The device is operated by the MinKNOW software that controls the device, experimental scripts, and performs basecalling.

Below is a quick start guide to set up a new device and to start sequencing your library samples. For further details using this device, please see the PromethION 2 Solo User Manual.

Install the P2 Solo with a GridION.

  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.

Install the P2 Solo with a workstation/laptop.

  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.

Log into MinKNOW.

  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.

Min login

Tutorials

These are available to help you get started and run your experiments as easily as possible.

When you open the MinKNOW software for the first time, the tutorials will navigate you through the graphical user interface (GUI). Click Continue tutorial to follow or click the to skip.

tutorial

Configure your GridION.

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.

Perform a hardware check.

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.

P2 hardware check

Perform a flow cell check.

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.

P2 flow cell check

Set up a sequencing run.

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.

start

7. Computer requirements

Firewall settings needed to run the MinION Mk1B/Mk1D

During runs both MinKNOW and the Desktop Agent require access to the AWS IP ranges currently listed here for the region eu-west-1, and also access to:

178.79.175.200

96.126.99.215

8. Installing MinKNOW on Windows

Download and installation

This part of the configuration will introduce the MinKNOW software and explain how to download and install it. There are three items to take particular note of:

  • If anti-virus software stops the installation, contact your IT department
  • The MinKNOW downloads as a .zip file and all files need to be extracted before proceeding with installation
  • Data generated from the MinKNOW software needs to be stored on the SSD drive, and care should be taken to ensure the data location is set to the SSD during installation

If help is needed during installation, please refer to the Support area in the Community.

Download MinKNOW

  1. Download MinKNOW software from the Downloads page on the Community
  2. For Windows, there are options for GPU and non-GPU metapackages. If you have a suitable NVIDIA GPU on your host system, you should use the GPU version of MinKNOW for significantly improved basecalling performance.
  3. Follow the on-screen instructions
  4. Contact the local IT administrators if there are any issues with permissions for installation

Extract the .zip files.

  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

Follow the installation prompts.

At the Installation Options screen, click 'Next' to accept default installation options.

In the Choose Data Location dialogue box, check that you are happy with the location of the sequencing reads data to change to the required SSD location and click on Install.

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.

Minknow installation 1a

Progression of installation

A dialogue box will open and shows progression of the installation.

Minknow installation 2a

When installation is complete, click on 'Close' to see MinKNOW successfully installed.

Navigate to the MinKNOW icon on the desktop.

END OF STEP

The presence of the MinKNOW icon on the desktop indicates that this step has been successfully completed.

OPTIONAL ACTION

If you have a proxy server and would like to set up MinKNOW using a proxy, follow the instructions below.

Open the user_conf file:

C:\ProgramFiles\OxfordNanopore\MinKNOW\conf\user_conf

And edit the following portion of the file:

"proxy": {
"cereal_class_version": 0,
"use_system_settings": true,
"auto_detect": true,
"auto_config_script": "",
"https_proxy": "",
"proxy_bypass": ""

Edit the https_proxy setting, which should be in the style of:

scheme://[username:password@]host:port or "http://domain\\username:password@host:port", where "scheme" is one of https, socks, socks4 or socks5.

9. Installing MinKNOW on Mac OS X

Download MinKNOW

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.

Apple install 1

3. Follow the on-screen instructions.

Apple install 2

4. Provide a fingerprint or password as required.

Apple install 3

Apple install 4

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

Navigate to the MinKNOW icon in the Applications folder.

END OF STEP

The presence of the MinKNOW icon in the Applications folder indicates that this step has been successfully completed.

OPTIONAL ACTION

Set the directory where the read files will be saved:

Open a command line and type:

sudo /Applications/MinKNOW.app/Contents/Resources/bin/config_editor --conf user --file /Applications/MinKNOW.app/Contents/Resources/conf/user_conf --set "output_dirs.base=/where the files will be saved"

Add the full path to the location where you would like the read files saved in the place of "where the read files will be saved".

OPTIONAL ACTION

If you have a proxy server and would like to set up MinKNOW using a proxy, follow the instructions below.

Open the user_conf file:

/Applications/MinKNOW.app/Contents/Resources/conf/user_conf

And edit the following portion of the file:

"proxy": {
"cereal_class_version": 0,
"use_system_settings": true,
"auto_detect": true,
"auto_config_script": "",
"https_proxy": "",
"proxy_bypass": ""

Edit the https_proxy setting, which should be in the style of:

scheme://[username:password@]host:port or "http://domain\\username:password@host:port", where "scheme" is one of https, socks, socks4 or socks5.

10. Installing MinKNOW on Linux

To add the Oxford Nanopore apt repository, run the command below on a terminal window:

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

Install MinKNOW using the command:

CPU version:

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

GPU version:
sudo apt update
sudo apt install ont-standalone-minknow-gpu-release

For GPU specifications, please refer to your sequencing device IT requirements document.

Open the MinKNOW GUI from the applications list.

Default installation directories

For the MinKNOW software: /opt/ont/minknow

For the MinKNOW user interface: /opt/ont/minknow-ui

Location of the reads folder:

The reads folder is in /var/lib/minknow/data

Location of the log files:

The MinKNOW logs are located in /var/log/minknow The Dorado basecaller logs are located in /var/log/dorado

OPTIONAL ACTION

If you have a proxy server and would like to set up MinKNOW using a proxy, follow the instructions below.

Open the user_conf file:

/opt/ONT/MinKNOW/conf/user_conf

And edit the following portion of the file:

"proxy": {
"cereal_class_version": 0,
"use_system_settings": true,
"auto_detect": true,
"auto_config_script": "",
"https_proxy": "",
"proxy_bypass": ""

Edit the https_proxy setting, which should be in the style of:

scheme://[username:password@]host:port or "http://domain\\username:password@host:port", where "scheme" is one of https, socks, socks4 or socks5.

11. Updating MinKNOW for Windows and Mac OS X

MinKNOW updates

Availability of updates to the MinKNOW software are indicated via the MinKNOW host settings or as a pop-up when first opening the software. The user should follow the on-screen instructions to install the new versions.

The details of the update will be communicated in Nanopore Community announcements.

We strongly recommend users to update as soon as reasonably possible after the release has been made available.

Note: Users will not be able to update their device if connected remotely or if a run is in progress.

IMPORTANT

Securing custom scripts prior to updating MinKNOW

If you have created custom scripts in MinKNOW, care should be taken to store them securely so they are not overwritten during software updates.

Please also be aware that the script structure can change between MinKNOW versions; custom scripts written for one version may not be compatible with the next.

Open the MinKNOW UI via the desktop icon and the auto-updater will automatically check for updates when connected to the device.

A dialogue box will open when a new update is available.

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.

36

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.

You can also update the device from the Software page of the Host settings.

IMPORTANT

Re-installing MinKNOW

In some cases (e.g. if there is a problem with the version of MinKNOW that you are using), it may be necessary to re-install the software. To do this, first do a full uninstall of the current version by following the instructions in the Support article: How to uninstall MinKNOW Software for the MinION Mk1B, Mk1D and P2 Solo Devices.

12. Updating MinKNOW for Linux

To update MinKNOW to a new version:

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
IMPORTANT

Re-installing MinKNOW

In some cases (e.g. if there is a problem with the version of MinKNOW that you are using), it may be necessary to re-install the software. To do this, first do a full uninstall of the current version by following the instructions in the Support article: How to uninstall MinKNOW Software for the MinION Mk1B, Mk1D and P2 Solo Devices.

13. Login and logout

We recommend users to log into the MinKNOW software using their Community credentials.

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.

Login

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

Okta 2 email address login page

Okta 3 password page external users

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

To log out of the MinKNOW software:

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

Picture24

14. Connection Manager

The Connection Manager enables you to connect to your devices.

MinKNOW can be accessed directly on the device or from another computer that remotely connects to the device used for sequencing, and summarises the states of the flow cells in the device.

  • Blue tag: direct connection
  • Purple tag: remote connection

Click Add host and enter the IP or hostname of a device to add it to the connection manager for remote access.

Screenshot 2023-03-22 172315

To interact with a device, click on the device you want to access from the Connection Manager.

You will be navigated to the Sequencing Overview page of the device where the flow cell states are displayed.

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

You can now navigate through MinKNOW, start a new sequencing experiment, perform post-run analysis, or view previously run experiments on the device.

15. Device security settings

Default security settings

Default security settings for devices requires users to have a Nanopore account to remotely connect to a device.

To change the remote access availability, settings can be updated when directly connected to a device.

Open 'Host settings' when directly connected to a device.

Navigate to the 'Device settings'. Security settings options are located in 'Remote access availability'.

Select options for local and remote access.

Screenshot from 2021-10-29 15-49-46

16. Homepage

Homepage overview

The MinKNOW Homepage enables you to navigate to:

a. The Start homepage b. Sequencing Overview of connected flow cells c. Recent and current Experiments d. System Messages e. Host Settings f. Connection Manager to connect with other available devices g. Start Sequencing experiment h. Post-run Analysis i. Flow Cell Check j. Hardware Check k. More includes option to generate .mmi from .fasta file or to import a sample sheet l. Guest/initials to logout

Annotated first page

Sequencing Overview

This page displays the inserted flow cell state and progress of a current sequencing experiment, including pausing, pore scan and basecalling.

Flow cell with no checks: This example is on a GridION device. Seq overview no check 1

Sequencing states: These examples are on a GridION device.

  • Pore scan: Pore scan 1

  • Flow cell health after the first pore scan: sequencing 1

  • Run paused: pause 1

  • Basecalling catch up post-sequencing: Basecalling 1

Click the flow cell to open the quick view of the current experiment. 3

Experiments page overview

The experiments page displays summary information for all experiments and device check data. You can control runs and see real-time information from sequencing flow cells.

All previous runs can be viewed here by altering the number of days available in "Experiments active in the last ... days".

experiment

System Messages

All device reports and messages are displayed here.

Picture25

17. Hardware check

IMPORTANT

Hardware check

A hardware check must be performed on all new devices or when software has been updated. This uses the Configuration Test Cell(s) (CTC), that come pre-inserted into your device in place of flow cells.

Note: If using a Flongle Flow Cell, we recommend regularly checking the Flongle adapter by inserting the empty adapter and completing a hardware check. For checking the device or flow cell position, use a MinION CTC, even if a Flongle Flow Cell will be used for sequencing.

Insert a Configuration Test Cell (CTC) into the device.

Navigate to the start homepage and click 'Hardware check'.

Hardware check 1

Click 'Start' for the check to begin.

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. hardware flongle


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

You will be automatically navigated to the Sequencing Overview page.

A loading bar will be displayed under the flow cell during the checks.

This example uses a MinION CTC. Hardware check

END OF STEP

The hardware check will complete after approximately one minute.

Hardware check results are indicated as follows in the top left corner:

  • Pass is indicated by a green check icon
  • Fail is indicated by an orange check icon

hardware check output

If the hardware check fails, remove and reinsert the CTC, and run a hardware check again. If the check fails for a second time, please contact Technical Support via email (support@nanoporetech.com) or via Live Support in the Nanopore Community.

18. Flow cell check

Flow cell check

We strongly recommend performing a flow cell check before loading a DNA or RNA library to assess the number of pores available.

Oxford Nanopore Technologies will replace any flow cell that falls below the warranty number of active pores within 12 weeks of purchase, provided that you report the results within two days of performing the flow cell check and you have followed the storage recommendations.

Flow cell Minimum number of active pores covered by warranty
Flongle Flow Cell (FLO-FLG001) 50
MinION/GridION Flow Cell 800
PromethION Flow Cell 5000

Navigate to the start homepage and click 'Flow cell check'.

Flow cell check

When you see the flow cell type and the flow cell ID is recognised, click 'Start' to begin.

This example is on a GridION device:

Note: For Flongle, the flow cell ID must be filled in manually. Flow cell check start 1

IMPORTANT

For Flongle, flow cell ID is not automatically assigned in MinKNOW. It is imperative the flow cell ID is entered in the correct format: ABC123 i.e. ([A-Z] x 3 [0-9] x 3).

The ID is case sensitive with no spaces. The MinKNOW software will not allow you to proceed until the flow cell ID has been entered correctly.

The user will be notified in the bottom right corner of the GUI if any information entered is in the wrong format or missing.

The Flongle flow cell ID is shown in the blue box below:

Flongle flow cell ID

You will be automatically navigated to the Sequencing Overview page.

During a flow cell check, a loading bar will appear underneath the flow cell to indicate the progress of bringing the flow cell up to temperature before another loading bar appears for the flow cell checking process.

This example is a MinION Flow Cell. Flow cell check

Flow cell health indicators

The quality of the flow cell will be shown as one of the three outcomes on the Sequencing Overview page:

  1. Yellow exclamation mark: The number of sequencing pores is below warranty.

  2. Green tick: The number of sequencing pores is above warranty and ready for sequencing.

  3. Question mark: A flow cell check has not been run on the flow cell during this MinKNOW session.

flow cell check indicators

Note: the indicator of quality will only remain visible during the MinKNOW session when testing occurred. Once the sequencing device has been re-started, the status of the flow cell will be erased.

19. Experiments page

Experiments page

The experiments page displays summary information for all sequencing flow cells and device checks carried out on the device.

Previous runs can be viewed on the UI at the top of the screen and the number of days to view the last active experiments can be altered by typing in a different integer. Altering days of experiments

From this page, you can control specific runs and identify real-time information, including flow cell health and reads.

  • Run statistics: The total number of reads, estimated and basecalled bases across an experiment, and number of active and total runs
  • Run time: The duration of the experiment
  • Run state: The current state of the sequencing run; 'Active', 'Basecalling', 'Complete', 'Stopped with error'
  • Health: The current flow cell health

The white panel displays a summary of sequencing experiments and the blue panel displays status information of a specific run.

Example of experiments page on GridION: experiment 1

For more status information of a specific run, click the run to open the quick view, including current temperature and voltage. In the example below, the run in position X3 was clicked to open the quick view. experiment 2

Live sequencing graph configuration

Page configuration allows you to choose which graphs to generate in the quick view of an experiment.

To open page configuration, click on a run in the white panel to open the quick view and select the highlighted button to choose which graphs to display.

page config

Tabs:

  • Default: Default graphs available
  • All: All graphs available
  • Custom: Users can move graphs from 'Available pages' to 'Your pages' to display a custom order of graphs in the GUI. On this page, you can also choose graph order. Click the graph and drag, then click Save.

Page configuration custom

Graphs

Click on the specific run to open the experiment details and view the graphs. Use the arrows to navigate between the graphs.

graphs

For more information about the graphs, refer to Check and monitoring in the 'Monitoring and troubleshooting your sequencing run' section.

Pausing

Sequencing experiments can be paused by selecting and pausing a single or all flow cell positions.

Pausing works by dropping the voltage potential over the membrane to 0 mV to maintain a safe environment to add components, such as more DNA/RNA library or nucleases for a flow cell wash. Data acquisition will continue during this period, as this prevents sequencing data from being lost.

To pause, click Pause in the run controls to open a dialogue box and select which flow cell position(s) to pause and click Pause.

pausing

Triggering a pore scan

The pore scan is used to assess the quality of the four wells in each channel to select the best performing pores. A new pore scan can be triggered every time a sequencing experiment is resumed after a pause (e.g. for a flow cell wash), or if the number of sequencing pores has significantly dropped during an experiment.

Navigate to the Experiments page, open a specific run, and click Start pore scan in the run controls. Confirm the flow cell to be scanned and click Start pore scan.

pore scan 2

20. Save sequencing run settings

Sequencing run template

Sequencing parameters can be saved as a template in MinKNOW at the review stage for users who regularly use the same parameters and to save set-up time. The template can be loaded when setting up a new sequencing run but can be reviewed and edited before starting the run.

Set up a sequencing run on MinKNOW as outlined in the "Starting a sequencing run" section of this protocol.

On the review tab, click 'Save settings as template' before starting the sequencing run.

Run config Save settings 6 0

Fill in the template name and click save.

Run config Save settings Select save 6 0

To use the saved template on a new sequencing run, click 'Start sequencing' and fill in the experiment name and select the flow cell position(s).

Save settings 12

Click 'Load settings from template' and select the template to use. Click load to open the template.

Saved settings can also be deleted from this window by clicking Delete.

Save settings 13

Confirm the kit and flow cell type to load by clicking 'Yes' and fill in the sample ID(s).

Save settings 6

Once the template has loaded, fill in the sample ID(s).

Review the sequencing parameters on the review tab and start the run by clicking 'Start'.

Run config Save settings RUN 6 0

Users can also join an existing experiment that has been previously run on the device:

  1. Fill in the experiment name and select flow cell position.
  2. Choose the same flow cell type as used in the previous experiment.
  3. Click Join existing.
  4. Select the previous experiment to run and click Join.
  5. The previous experiment parameters will load with the experiment name.
  6. Fill in the sample ID(s) and review settings in the "Review" tab and start the run.

Note: Flow cell types must be the same between both experiments to join.

Save settings 11

21. Sample sheet upload

For GridION and PromethION experiments, or when multiple MinION Mk1B/Mk1D or MinION Mk1C devices are running simultaneously, it may be preferred to upload sample names and corresponding flow cell positions from a CSV file, rather than manually.

The sample sheet may describe flow cells being run at one or more than one position. The columns available are as follows, though some are optional:

Column title Description Notes
flow_cell_id Defines the flow cell ID which applies to the sample sheet row. Used to identify which positions to apply values to in MinKNOW.
This is optional if position_id is specified
position_id Defines the flow cell position which applies to the sample sheet row. Used to identify which position to apply values to in MinKNOW.
This is optional if flow_cell_id is specified.
sample_id Defines the sample ID to be applied in the run An individual position can only have one sample_id assigned to it when starting a run.
This is optional.
experiment_id Defines the experiment ID to be applied Each row in the sample sheet must contain the same experiment_id value for the sample sheet to only have a single experiment_id value defined overall. All entries in each row will be validated.
flow_cell_product_code Defines the product code of the flow cell Used to find the correct sequencing script to start the run
kit Defines the kit and any expansion kits used with the sample Used to find the correct sequencing script to start the run. The sample sheet must contain only one sequencing kit, but expansion kits are not limited. If expansion kits are additionally defined, they should be separated by a space: e.g. SQK-LSK109 EXP-NBD104 EXP-NBD114
Note: Ensure the correct format of the kit name is used, converting any full stops in the kit name to a dash. For example, the Rapid Barcoding Kit V14 96 (SQK-RBK114.96) format in the sample sheet would be SQK-RBK114-96.

For experiments that involve barcoding, an alias is associated with each barcode (or pair of barcodes where appropriate). Additional columns are available for barcoding runs:

Column title Description Notes
alias User-specified string which applies a given label to a specific barcode or barcode pair. The alias cannot be an existing barcode folder name e.g. 'unclassified', 'classified' and 'mixed' are not allowed. The alias must be between 1 and 40 characters. The character set needs to match the following format: /^([0-9a-zA-Z-_] and only contain the following characters: numbers, upper/lower-case letters, dashes, and underscores.
type One of the options: test_sample, positive_control, negative_control, no_template_control This is optional.

One or two columns define the barcode or barcode pair. The exact form is dependent on whether single or dual barcoding is being used.

Single barcoding: the sample sheet contains one barcoding arrangement column:

Column title Description Notes
barcode The barcode identifier for the row e.g. barcode01

Dual barcoding: the sample sheet contains two barcoding arrangement columns:

Column title Description Notes
internal_barcode The internal barcode identifier for the row, e.g. internal01.
external_barcode The external barcode identifier for the row, e.g. external01.

Column titles are defined within the first row of the sample sheet. These must be defined in lowercase using the column title values listed above.

Sample sheet validation occurs against the hardware and between rows in the sample sheet to ensure validity when the sample sheet is loaded. Validation and importing the sample sheet requires all the flow cells to be used in the experiment. Be careful to ensure the correct flow cells and positions are used.

Below is an example sample sheet CSV file for using the Native Barcoding Expansion 96 (EXP-NBD196) with the Ligation Sequencing Kit (SQK-LSK109) on a GridION device for a single position.

Sample sheet recommendations

Sequencing parameters using a JSON file

Alongside a CSV file containing the sample information, users can also upload a JSON file containing the sequencing parameters for their experiments rather than inputting the information manually.

The JSON file is exported from MinKNOW and contains all default sequencing parameters that can be altered:

Run options tab:

  • "runLengthHours": 72 (Run length in hours condition. This must be an integer.)
  • "runLengthCondition": available_pores (Run length based on flow cell end of life condition. Run length in hours will be ignored if this condition is set.)
  • "targetDataCondition": estimated_bases or passed_basecalled_bases (Run length based on target data output. This can be data estimated or data basecalled, respectively.)
  • "enrichDepleteAdaptiveSamplingEnabled": false,
  • "enrichDepleteAdaptiveSamplingRefFile": null,
  • "adaptiveSamplingChannelStart": 1,
  • "adaptiveSamplingChannelEnd": 512,
  • "enrichDepleteAdaptiveSamplingBedFile": null,
  • "shouldEnrichAdaptiveSamplingRef": true,
  • "barcodeBalancingEnabled": false,
  • "barcodeBalancingCustomBarcodes": false,
  • "barcodeBalancingBarcodeSelection": null,
  • "muxScanPeriod": 1.5,
  • "reservedPores": true,

Analysis tab:

  • "basecallingEnabled": true, (Basecalling can be switched on/off as true/false)
  • "modifiedBasecallingEnabled": false, (Modified basecalling can be switched on/off as true/false. To enable methylation calling, this needs to be enabled.)
  • "barcodingEnabled": true, (Barcoding can be switched on/off as true/false)
  • "hasCustomBarcodesSelection": false,(This option can be switched on/off as true/false)
  • "customBarcodesSelection": null,
  • "basecallModel": "dna_r9.4.1_450bps_hac.cfg", (Basecalling model depends on the flow cell and kit combinations. To enable methylation calling, this needs to be enabled.)
  • "modifiedBasecallingContext": "", (To enable methylation calling, this needs to be enabled)
  • "trimBarcodesEnabled": true, (Trim barcodes can be switched on/off as true/false)
  • "requireBarcodesBothEnds": false, (Requirement to have barcodes on both ends can be switched on/off as true/false)
  • "alignmentEnabled": false, (Alignment option can be switched on/off as true/false)
  • "alignmentRefFile": null,
  • "alignmentBedFile": null,
  • "workflowEnabled": false, (EPI2ME workflows can be switched on/off as true/false)

Output tab:

  • "offloadOrOutputLocationNavParams": "path": "/data/."
  • "fastQEnabled": true, (FASTQ files can be switched on/off as true/false)
  • "outputSplitTypeFormat", (Split reads by time or by file number with either of the following arguments)
    • "time_elapsed"
    • "basecalledOutputPeriod" : 600 (Time between file chunks, in seconds. This is an integer)
  • "number_of_reads", "outputReadsThreshold" : 4000 (Number of reads per file. This is an integer)
  • "fastQDataCompression": true, (FASTQ compression can be switched on/off as true/false)
  • "fast5Enabled": true, (FAST5 files can be switched on/off as true/false)
  • "fast5DataCompression": "vbz_compress", (Compression type. This can be "vbz_compress" or "zlib_compress")
  • "pod5Enabled": false,
  • "splitPod5FilesByBarcode": false(Split raw read files by barcode can be switched on/off as true/false)
  • "bamEnabled": false, (.BAM output can be switched on/off as true/false. To enable methylation calling, this needs to be enabled.)
  • "bamWriteMultiple": true,
  • `"readFilteringEnabled": true,
  • `"readFilteringMinQscore": 9,
  • "readFilteringMinReadlength": null, (read length in kb)
  • "readFilteringMaxReadlength": null, (read length in kb)
  • "readSplittingEnabled": false,
  • "overrideMinReadSplittingScore": false,
  • "minReadSplittingScore": 58,
  • "bulkFileEnabled": false,
  • "bulkFileRaw": "1-512",
  • "bulkFileEvents": "1-512",
  • "bulkFileReadTable": "1-512",
  • "bulkFileRawEnabled": false,
  • "bulkFileEventsEnabled": true,
  • "bulkFileReadTableEnabled": true,

Navigate to the Start page.

Click 'More' and click 'Sample sheet import'.

1

Select and upload the CSV file.

2

Click 'Export run settings as template' when the CSV file has been uploaded.

Sample sheet edit

Open the exported JSON file to edit sequencing parameters, as described above and save.

Upload the JSON file by clicking 'Import run settings'.

4

Click 'Start' to start the run using the CSV and JSON files.

Error messages that you may see if you make a mistake in the sample sheet:

  • Position defined using position_id does not exist
  • Position defined using flow_cell_id cannot be found
  • flow_cell_id and position_id are used but the flow_cell_id does not match the flow cell used
  • flow_cell_product_code is used but the value doesn't match the flow cell used
  • Different sample_id values are assigned against the same position whilst setting up a run
  • flow_cell_product_code and kit do not match
  • Zero or more than one sequencing kit listed in kit
  • Barcode kits in kit do not exist as an option on the selected protocol
  • type must be a valid selection from the list of valid types outlined above
  • experiment_id must exist within each row and must all have the same value

22. Application settings

Application settings contains the tutorials, account/login settings and MinKNOW GUI information.

Navigate to application settings from clicking the initials in the top right corner of the GUI.

The Application settings are available on the Connection manager as well as the device homepage.

Below is an example from the Connection manager: application settings

Tutorials

Tutorials are available here; they are also presented when logging into MinKNOW for the first time. You can use Recap to view a specific tutorial or use Reset tutorial state to go through all the tutorials again on opening the UI.

Tutorials

Account settings

Navigate here to logout from MinKNOW.

User email address used for login is displayed here, unless logged in as a guest.

account settings edit

App information

The installed version of the MinKNOW GUI is available here.

Click Reload to refresh the GUI.

App info

Language settings

To select your language, navigate to the language tab:

MinKNOW UI Language select 5.9

23. Overview

Features of the Host settings

The Host settings have several features to co-ordinate the device. From this area, users are provided with information on system parameters, such as storage, date/time and IP address.

To navigate to this area, click 'Host settings' in the side panel.

Picture3

Use the side panel to navigate through the host settings option. Depending on the device, different options are available. Below is a full list of the options available in the host settings:

Device Settings: The devices can be shut down and rebooted from this page, and settings for the time and date can be altered. Disk space can be managed and peripherals may also be added and ejected from here. Software: Users are able to download the latest software updates from this page. The option to update will only appear when an update is available. File Manager: Stored data on the device can be managed and transferred. System: Overview showing disk space utilisation, CPU and memory usage. Help: Device logs can be exported using the Export Logs function. Logs and temporary data can also be cleared by using the Clean up function. If troubleshooting of the device is required, a Repair problems function is available as well as a MinKNOW restart option. Network settings: Ethernet and WiFi settings are available here with the option to hotspot the device to connect to a larger screen. API access Beta features: An overview of the beta features available on the device. Either individual or all beta functions can be disabled from here.

Click Back to Main Menu to leave host settings.

Picture5

Host settings pages

  • Device settings: Unavailable on MinION Mk1B/Mk1D.

View from remotely accessed device: Picture6

View directly accessed device: direct access

  • Software: Available on all devices. Picture7

  • File manager: Unavailable on MinION Mk1B/Mk1D. Picture8

  • System manager: Unavailable on MinION Mk1B/Mk1D. Picture9

  • Help: Available on all devices. Picture10

  • Network settings: Available on MinION Mk1C and MinIT. Picture11

  • Beta features: Available on all device. Picture12

24. Shutting down

Navigate to the Device settings and click "Shut down".

Click Shut Down in the pop-up to confirm.

shut down

25. Rebooting

Navigate to Device settings and click "Reboot".

Click "Reboot" in the pop up to confirm.

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

reboot

To restart MinKNOW on GridION, navigate to 'Help' in the host settings and click "Restart MinKNOW".

Restart

26. System package updates

System package updates

System package updates for your device can be performed through the MinKNOW UI.
Availability of system package updates are indicated via the MinKNOW host settings in the software tab.

System package updates will not affect sequencing accuracy or quality.

We recommend maintaining your device updated with the the most up-to-date system packages.
However, having out-of-date system packages will not impact sequencing accuracy or quality.

Perform system package updates at your discretion and in accordance with your IT policies.

Note: Users will not be able to update their device if connected remotely or if a run is in progress.

Navigate to the software tab in host settings.

Software tab navigation MinKNOW

In the System window, click "Install packages".

Software tab install page System Pack install

The system package update can be monitored via the progress bar.

MinKNOW system update progress

Once the update completes successfully, power cycle your device to complete installation.

MinKNOW system update reboot

TIP

In event of a system package update failiure, an error message will appear.

The error message contains copyable details that you can use to assist with tech support.

MinKNOW system update error

If you see some system packages remain after a system update, please update MinKNOW and the number should return to zero.

System package update leftover packages

For more information on updating MinKNOW visit the Updating MinKNOW for Windows and Mac OS X or Updating MinKNOW for Linux sections of this document.

27. Data management

File manager

Data can be managed and transferred from the file manager tab on the host settings.

Navigate through the tabs to view the data stored:

  • Internal tab: Data stored on the connected sequencing device (MinION Mk1C, GridION, PromethION)
  • Removable tab: Data stored on a connected removable storage device e.g. USB drive
  • Network tab: Data stored on a connected network drive. The network drive must be mounted prior.

HS 6

To remotely access the device as a shared/network drive:

Navigate to device settings and switch the Share toggle on.

Picture16

A new window will open to create a password for security.

Picture17

Click Set password, and after a few seconds the share toggle will be active.

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

image (26)

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.

TIP

To stop sharing, click the toggle to turn off.

Note: The password will be removed and must be reset when sharing is turned back on.

To manage data between the internal, removable and network tabs:

Switch the Select toggle on to open the greyed out options Copy, Move, Delete or Rename in the bottom right corner.

Picture18

Select a folder or file to use the options in the bottom right corner.

Picture19

Click either Copy or Move to open a new dialogue box with a list of all the drives accessible to the device.

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

Picture20

Click on a drive to open and navigate through the file directory to choose destination. Click either Copy or Move to confirm action.

To use the delete function, select a file or folder, and click "Delete" and confirm action in the pop up.

Picture22

To use the rename function, select a file or folder, and click "Rename" and type in the new name and confirm in the pop up.

Picture23

Compatible disk formats:

SD and USB on MinION Mk1C supports DOS FAT, Windows 95 FAT, xFAT, and Linux ext(2|3|4).

New directories can be created from the device GUI using the New Directory icon.

Click the icon in the bottom right corner to open Create New Directory and type in a directory name. Click Create.

Picture24

Disk management

Navigate to Device Settings to view available disk space on the device. Any peripherals plugged in, including USB, can be ejected using the Eject option.

A network drive can also be connected to the device by selecting Add Network Drive and filling in the required credentials for connecting using a Samba (SMB) server or a Network File System (NFS).

device settings

28. Mount Network Drive

Network drives

Network drives for data storage can be mounted from either the Device Settings or the File Manager in the Network tab:

  • Device Settings: Picture1

  • File Manager: Picture2

Click "Add network drive".

A modal will open. Click either SMB (Samba) or NFS (Network File System) shares.

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: HS 2

NFS: HS 3

To mount SMB shares:

  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.

Mount network drive 3

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

To mount NFS shares:

  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.

Mount network drive 4

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

29. Tutorials

Tutorials

On opening the MinKNOW software, the tutorials will start to navigate you through the user interface.

Picture26

Skip tutorials

Tutorials may be skipped if you are familiar with the user interface.

To skip tutorials, select the three dots and choose to either skip all tutorials or just a section.

Skip tutorials

Review tutorials

Tutorials can be reviewed again by selecting Reset tutorial state in the Tutorials panel of Application Settings.

Review tutorials

30. Network settings and connection

Network settings display internet connectivity information. The MinION Mk1C can be connected via Ethernet or Wi-Fi.

Both options are supported, however if Ethernet is available, we recommend using Ethernet because the connection will likely be quicker and more reliable.

Ethernet

To connect using Ethernet:

  1. Connect a Ethernet cable to the device
  2. Navigate to network settings in host settings to confirm connection. IP configuration information will appear when connected.

Mk1c 1

Wi-Fi

To connect using Wi-Fi, navigate to network settings and toggle Wi-Fi on. Select a network from the Available Networks panel to connect to the internet.

Network settings 2

Select Advanced to open Advanced Wi-Fi Configurations. From here, all available networks can be viewed, connected or removed.

Network settings 4

IMPORTANT

Please note that Wi-Fi connections with captive portals (a web page that the user is required to view and enter login details before access is granted) cannot be used with the MinION Mk1C.

Configuring static IP address

The static IP address can be configured through MinKNOW UI for both WiFI and Ethernet. Below is an example for Ethernet.

  1. Click Edit in "IP configuration" to open "Edit the IP address configuration" dialogue box. Mk1c 1
  2. Select either DHCP or Static tab and click Confirm to save. Mk1c 3
  3. Once the IP address configuration has changed successfully, click Finish to close the dialogue box. Mk1c 4

Hotspot can be activated to allow users to connect to a larger screen.

This can be used to connect to the MinION Mk1C in places where there is no network infrastructure, e.g. in the field. To enable hotspot, select the toggle to ON. When active, the device hotspot name will be displayed.

image (62)

Hotspot configs can be edited to select a channel if required for congested networks where there may be competition for channels.

  1. Navigate to the network settings
  2. For hotspot, select the toggle to ON
  3. Click 'Configure hotspot'
  4. Select channel using the dropdown menu
  5. Enter new password
  6. Click 'Confirm'

The hotspot should be confirmed as specified in the hotspot configs.

wifi hotspot mk1c

To connect the MinION Mk1C Wi-Fi router to a Windows device:

  1. Locate the MinION Mk1C ID on the underside label of the device:

MinION Mk1C serial label

  1. Click on the MinION Mk1C ID under the Wi-Fi Connections Panel in the Control Panel or the options in the right side of the Task Bar

Login with the Security Key: WarmButterflyWings98

mc-110016 Wifi ID

  1. Access the MinION Mk1C by the File Explorer: Go to \\mc-XXXXXX (XXXXXX is the MinION Mk1C ID) in the Address Bar. You will be prompted for the network credentials on connecting to the MinION Mk1C through File Explorer.

To connect the MinION Mk1C router to a macOS device:

  1. Locate the MinION Mk1C ID on the underside label of the device:

MinION Mk1C serial label

  1. Click on the Wi-Fi icon on the right of Menu bar at the top of the screen:

Wi-Fi options white border 3.0 smaller

Identify the MC-XXXXXX ID in the Network Name under Network Panel (the final option under the Wi-Fi icon), which is show in the window below (left)‡. Selecting the MinION Mk1C ID and clicking Apply will present the Wi-Fi login pop-up. Use the password credentials shown below.

Login with the Password: WarmButterflyWings98

wifi log in for macOS

  1. Access the MinION Mk1C by the Network browser: Search for Network using Finder. Click on Server in the tab to lower left of the Search bar.

MC-XXXXXX (XXXXXX is the MinION Mk1C ID) will be displayed as a server option. Click the MC-XXXXXX icon, click Connect as under the Search bar and enter the network credentials.

connect as MAC

‡ When connecting to your device by SSH, it can be addressed in three different ways:

  1. Using DNS
  2. Using mDNS
  3. Using its IP address directly

In most organisations, central DNS servers are used. If your device is using a 'static' IP configuration, your IT department will have to create a DNS entry for it manually. If your device is using a dynamic (DHCP) IP configuration, the DNS entry will often be created automatically. To SSH into your device using DNS, set the hostname in your client to the device's hostname, e.g. MC-112345.

If DNS is not provided by your organisation or your broadband router, try using mDNS. When your device is connected to a network, it announces its presence to all other nearby devices such as your laptop or PC. If your laptop or PC is configured to recognise these announcements, you can SSH into your device using mDNS by setting the hostname in your client to the device's hostname followed by ".local", e.g. MC-112345.local.

If the above methods are not successful, use the devices IP address directly. The MinKNOW UI shows the IP addresses for each interface in the Settings - Network page. If your device has LAN and Wi-Fi interfaces, each will have a different IP address. To SSH into your device using its IP address, set the hostname in your client to the IP address, e.g. 10.20.30.41.

To connect the MinION Mk1C router to a Linux device:

  1. Locate the MinION Mk1C ID on the underside label of the device:

MinION Mk1C serial label

  1. Click on the Wi-Fi icon on the right of Menu bar at the top of the screen.

Wi-Fi options for Linux 3.0 white border

Identify and click on the MC-XXXXXX ID under the Wi-Fi network options icon (shown above). Enter the Wi-Fi password (shown below) in the pop-up Wi-Fi Network Authentication window:

Login with the Password: WarmButterflyWings98

Linux Wi-fi password - 2.0 white border

The following window will be presented when you have connected to the MinION Mk1C Wi-Fi router.

Wi-Fi connected for Linux 2.0 white border

  1. Access the MinION Mk1C using the Linux File Browser and enter the server address.

Open the File Browser (this can be found using the Search tool). Select Connect to server on the bottom of the list in the left panel. You will need to enter:

smb://mc-110013

31. Beta features

Beta feature availability

For devices with beta features available, users can choose to have beta features enabled or disabled on their device. By default, these features will be enabled and highlighted in the UI with a yellow "beta" badge.

To disable the beta features:

  1. Navigate to Beta features in the host setting.
  2. To disable all beta features and enable the "Beta features enabled" switch.
  3. To disable an individual feature, uncheck the tick box for the for the specific beta feature.

beta features

32. Troubleshooting your device

If there are any issues associated with your device or sequencing, please contact Technical Services.

To find our most common FAQs, please see the support section for MinKNOW here.

In most cases where troubleshooting is required, we recommend exporting your logs and sending them to Technical Services.

Help feature and support link from MinKNOW.

The Nanopore support hub can also be accessed directly from your MinKNOW UI.

In the top right of MinKNOW you will see the "Help" option:

MinKNOW UI Help option 6 0

Clicking this option will bring you to a page with a link and a QR code:

MinKNOW UI Help option QR 6 0

On larger devices such as the GridION and PromethION 24/48, and devices such as the MinION Mk1B/Mk1D and P2 Solo, clicking the link will directly bring you to the Nanopore support page.

On devices such as the P2 Integrated and MinION Mk1C, you will be able to scan the QR code with another device, or type in the shortened URL on another device to quickly reach the support hub.

To export logs:

For the PromethION 24/48, PromethION 2i, GridION and PromethION 2 Solo connected to a GridION:

  • Click Export in the Export Logs section.

When you have successfully exported the logs, you will be notified of where they are located below the button. The logs will be downloaded as a TGZ file in the logs directory.

You can change the location of the logs by using the file manager, where you can easily retrieve them without needing SSH:

  • Click on the file next to Logs exported to: in the Export Logs panel. This will open where the logs are located in the File Manager of MinKNOW.
  • Use the control panel in the lower right corner of the File Manager to move the log files.

export logs

If the PromethION 2 Solo is connected to a stand-alone computer

Log files for each sequencing experiment can be found in:

  • Windows C:\data\logs

  • Mac OS X /private/var/log/MinKNOW

  • Linux /var/log/minknow

To remove logs and temporary data:

In the "Help" page, click Clean up in the Clean Up Files section and confirm in the in the pop-up.

Note: Run data will not be deleted. Logs and temporary data will be removed.

clean up

To repair device issues:

Click Run repair in the troubleshooting section of the Help page and confirm in the pop-up. MinKNOW will attempt to repair the following issues:

  • Ensure required directories under "/data" exist and have the correct ownership and permissions
  • Ensure required files under "/data" have the correct ownership and permissions
  • Ensure the GPU is working at maximum performance
  • Ensure that Bluetooth is disabled

Screenshot 2023-01-24 125625

33. Introduction to local basecalling

Introduction to live basecalling in MinKNOW

For MinION Mk1B/Mk1D, Flongle on MinION Mk1B/Mk1D, and PromethION 2 Solo, the MinKNOW software presents an option to basecall reads on the local computer. The basecalling is carried out live, as the read files are generated during a sequencing experiment.

Basecalling results are displayed in real-time in the MinKNOW user interface, and data is written out in the BAM or FASTQ file format.

MinKNOW 3

Fast, High Accuracy and Super Accurate models and compatibilities

The MinKNOW basecallers offer three different basecalling models: a Fast model, a High accuracy (HAC) model, and Super accurate (SUP) model.

The Fast model is designed to keep up with data generation on Oxford Nanopore devices (MinION Mk1C, GridION, PromethION). The HAC model provides a higher raw read accuracy than the Fast model and is more computationally-intensive. The Super accurate model has an even higher raw read accuracy, and is even more intensive than the HAC model.

For more information about basecalling accuracy, see the Accuracy page on the Oxford Nanopore website.

A comparison of the speed of the models is provided in the table below:

2024 MinKNOW 6.0 basecall speeds 2

The number of keep-up flow cells assumes a 30 Gbase flow cell output in 72 hours for MinION and GridION, and 100 Gbase output in 72 hours for PromethION.

34. Starting a sequencing run with Flongle

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

Type in the experiment name, sample ID and choose flow cell type from the drop down menu. Fill in flow cell ID in the correct format: ABC123

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.

Flongle setup 1

IMPORTANT

For Flongle, flow cell ID is not automatically assigned in MinKNOW. It is imperative the flow cell ID is entered in the correct format: ABC123 i.e. ([A-Z] x 3 [0-9] x 3).

The ID is case sensitive with no spaces. The MinKNOW software will not allow you to proceed until the flow cell ID has been entered correctly.

The user will be notified in the bottom right corner of the GUI if any information entered is in the wrong format or missing.

The Flongle flow cell ID is shown in the blue box below:

Flongle flow cell ID

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

Flongle setup 2 experiment overview combined

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

step7

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

gen run report mk1b

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

35. Starting a sequencing run on MinION Mk1B/Mk1D

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

Type in the experiment name, sample ID and choose flow cell type from the drop down menu.

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.

MinION setup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

MinION setup 4 overview

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

step7

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

gen run report mk1b

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

36. Starting a sequencing run on MinION Mk1C

There are two versions of the MinKNOW GUI on the MinION Mk1C

The MinKNOW GUI on the device:

The version of the Graphical User Interface (GUI) described in this document is the touch-screen configurable GUI.

The MinKNOW GUI via Wi-Fi or Ethernet connection from a laptop:

The GUI presented on the laptop when connected to the MinION Mk1C is the same as MinION Mk1B/Mk1D desktop MinKNOW GUI. See the MinION Mk1C user guide for how to use the Wi-Fi or wired connection from a device to the MinION Mk1C version of the MinKNOW GUI.

Click the 'Start Sequencing' option on the Start homepage to choose the running parameters for your experiment.

start

Type in experiment name, sample ID and choose flow cell type from the drop down menu.

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.

Mk1C Steup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

MinION setup 4 overview

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

22

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment, click 'Stop' on the experiments page.

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

experiment overview

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.

stop1

Once sequencing is complete, generate a run report.

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.

run report

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

37. Starting a sequencing run on GridION

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

OPTIONAL ACTION

When running multiple flow cells simultaneously, it may be preferred to upload sample names and corresponding flow cell positions from a CSV file, rather than manually.

Please see Sample sheet upload for instructions.

Type in the experiment name and sample ID and choose flow cell type from the drop down menu.

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.

GridION setup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

GridION setup 2 overview combined

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

Grid sequencing

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

Grid once sequ

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

38. Starting a sequencing run on PromethION 24 and 48

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

OPTIONAL ACTION

When running multiple flow cells simultaneously, it may be preferred to upload sample names and corresponding flow cell positions from a CSV file, rather than manually.

Please see Sample sheet upload for instructions.

Type in the experiment name, sample ID and choose flow cell type from the drop down menu.

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.

Prom setup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

Prom setup 2 overview combined

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

Prom sequencing

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

Prom sequ

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

39. Starting a sequencing run on PromethION 2 Solo

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

OPTIONAL ACTION

When running multiple flow cells simultaneously, it may be preferred to upload sample names and corresponding flow cell positions from a CSV file, rather than manually.

Please see Sample sheet upload for instructions.

Type in the experiment name, sample ID and choose flow cell type from the drop down menu.

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.

P2Solo setup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

P2Solo setup 2 overview

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

p1 seq

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

Grid once sequ

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

40. Starting a sequencing run on PromethION P2i

Click 'Start Sequencing' on the Start page to set up the sequencing parameters for your experiment.

start

OPTIONAL ACTION

When running multiple flow cells simultaneously, it may be preferred to upload sample names and corresponding flow cell positions from a CSV file, rather than manually.

Please see Sample sheet upload for instructions.

Type in the experiment name, sample ID and choose flow cell type from the drop down menu.

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.

P2i setup 1

Select the kit and expansion(s) used to prepare the library.

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.

Setup 2

TIP

Default sequencing parameters

Click Continue to proceed to the run configuration page.

Default sequencing parameters will be pre-set.

For the majority of users and applications, we recommend keeping the default sequencing parameters unless you have a specific need to change them.

Experiment overview

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.

P2Solo setup 2 overview

Choose your basecalling options:

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.

Prom setup 3 basecalling

Prom setup 3 basecalling II

Basecall models

Basecalling models can be selected at two stages in the MinKNOW software:

  • Real-time basecalling: A basecalling model can be selected when setting up a sequencing run on the "Run configuration" page of MinKNOW. This enables basecalling of the sequencing run in real-time.
  • Post-run basecalling: Basecalling can be set up post-sequencing run on MinKNOW. Instructions can be found in the "Post-run basecalling" section of this protocol.

Dorado is the basecaller server in MinKNOW which provides multiple models for basecalling nanopore data.

There are three options for model selection in the drop down menu:

1. High-accuracy basecalling (HAC) - This is our default recommended basecalling model that provides a high single-molecule accuracy. It is currently 5-8 times more computationally-intensive than the Fast model, so users should ensure their data transfer and device utilisation is scaled appropriately for this.

2. Super-accurate basecalling (SUP) - The Super accurate model has an even higher single-molecule accuracy, and is ~3 times more intensive than the HAC model.

3. Fast basecalling - This model is able to keep up with a high-throughput sequencing experiment on a MinION, GridION or PromethION.

Note: GPU devices will basecall in near real-time; CPU devices, like a standard laptop for MinION Mk1B/Mk1D, will not maintain real-time basecalling.

Choose your barcoding options:

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

Prom setup 3 barcoding

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.

Prom setup 4 barcoding II

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.

Flongle setup 6 Barcoding II Both ends

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

Choose your alignment options:

By default, alignment is turned off.

Flongle setup 7 Alignment

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.

Flongle setup 7 Alignment II

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

Choose your adaptive sampling options:

By default, alignment is turned off.

Flongle setup 8 Adaptive sampling

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

Flongle setup 8 Adaptive sampling II

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

OPTIONAL ACTION

Advanced User Options

Flongle setup 9 Advanced sequencing options

Time between pore scans: The time between pore scans can be altered using this feature.

Reserved pore: The reserve pore feature prioritises consistency and accuracy over immediacy by reserving wells where voltages have dropped until later in the run, such that other wells can catch up. Switch off this feature to fully front-load sequence data acquisition.

Flongle setup 9 Advanced sequencing options II

Set up the data targets for your experiment:

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.

Flongle setup 10 data targets

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".

Flongle setup 10 data targets run limit

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.

Flongle setup 10 data targets data target

Select the output options:

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

Click the Edit icon to open the options.

Flongle setup 11 Output

Output settings:

Flongle setup 11 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.

Flongle setup 11 Output warning

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:

Based on time elapsed Based on number of reads
Basecalled files can be produced:
· every 1 minute (many small files)
· every 10 minutes (many small files)
· hourly (fewer larger files)
· at the end of the run (single large file).
Basecalled files can be produced for specified read thresholds (number of reads).

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.

TIP

Advanced User Options

Option to turn bulk file saving either on or off.

Note: This will result in much larger file sizes due to additional information about the run, which is used for debugging.

Advanced User Options 5.9 27Feb2024

Choose the filtering options:

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:

Flongle setup 12 Filtering

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

Flongle setup 12 Filtering options

Click 'Start' to run the experiment.

Flongle setup 13 Start run

You will be automatically navigated to the Sequencing Overview when sequencing starts.

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.

p1 seq

Click on the flow cell to open the quick view and check the number of active pores. The first pore scan should report a similar number of active pores (within 10-15%) to that reported in the flow cell check.

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.

To stop the experiment during sequencing, click "Stop" using the run controls on the experiments page.

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

step8

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.

stop1

Once sequencing is complete, generate a run report.

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.

Grid once sequ

END OF STEP

MinKNOW fallback folder

If writing out reads to your specified location fails (e.g. if the disk becomes full, or the connection is disrupted), the experiment will continue, and the reads will be written into a fallback folder:

Windows .POD5 or .fast5 files: C:\data\reads\fallback (with the standard run data name sample ID as a folder) FASTQ files: C:\data\reads\fastq_fallback (with the standard run data name sample ID as a folder)

Mac OS X: .POD5 or .fast5 files: /Library/MinKNOW/data/fallback FASTQ files: /Library/MinKNOW/data/fastq_fallback

Linux: .POD5 or .fast5 files: /var/lib/MinKNOW/data/fallback FASTQ files: /var/lib/MinKNOW/data/fastq_fallback

41. Refuelling your flow cell

IMPORTANT

Refuelling: general advice

Note: This is only relevant for our Kit 9 chemistry.

Refuelling is the replenishment of motor fuel in the sequencing experiment through the addition of Flush Buffer (FB) from the Flow Cell Priming Kit (EXP-FLP002). The translocation speed graph in MinKNOW can indicate whether it is necessary to top up fuel. Volumes and processes of refuelling are specific to the flow cell type. Please follow the instructions relevant to your flow cell type e.g. Flongle™, MinION™ or PromethION™.

When to refuel

If the DNA translocation speed drops below 300 bases per second, you may start to see a reduction in quality of data reflected in the Qscore. We therefore recommend topping up the flow cell with fuel, using the Flush Buffer (FB) from the Flow Cell Priming Kit. Please follow the instructions below if you wish to top up the fuel during an experiment.

TIP

Please consult the 'Priming and loading the [SpotON/Flongle/PromethION] flow cell' step from the relevant library preparation protocol for instructions on adding solutions to the flow cell type that will be used.

Refuelling a Flongle™ flow cell

  • Remove one tube of FB from the freezer and thaw by bringing to room temperature

  • Pause the experiment on the GridION position/on the MinION Mk1B/Mk1D on which the flow cell is being refuelled:

    a. Navigate to Experiments and open the experiment running b. Click Pause c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm

MinION Flongle

  • Pull back the seal tab to reveal the sample port
  • Pass 30 µl of FB through sample port
  • Reseal the tab, ensuring the drain ports are covered
  • Unpause the experiment on the relevant GridION position/on the MinION Mk1B/Mk1D by clicking Resume
  • (optional) Click Start pore scan to pick a new set of the best channels for the remainder of the sequencing experiment

Refuelling a MinION™/GridION™ flow cell

Instructions for refuelling a MinION/GridION flow cell:

  • Remove one tube of Flush Buffer (FB) from the freezer and thaw by bringing to room temperature

  • Pause the experiment on the GridION position/on the MinION Mk1B/Mk1D on which the flow cell is being refuelled:

    a. Navigate to Experiments and select the experiment running b. Click Pause c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm

Pausing

  • After opening the priming port, check for a small bubble under the cover. Draw back a small volume to remove the bubble (a few µl):

    a. Set a P1000 pipette to 200 µl b. Insert the tip into the priming port c. Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip

Visually check that there is continuous buffer from the priming port across the sensor array.

Complete the flow cell refuelling:

  • Load 250 µl of the FB into the flow cell via the priming port (i.e. not the SpotON port), avoiding the introduction of air bubbles
  • Close the priming port and replace the MinION Mk1B/Mk1D or GridION lid
  • Unpause the experiment on the relevant GridION position/on the MinION Mk1B/Mk1D by clicking Resume
  • (optional) Click Start pore scan and choose your flow cell, to pick a new set of the best channels for the remainder of the sequencing experiment Pore scan

Refuelling a PromethION™ flow cell

  • Remove one tube of FB from the freezer and thaw by bringing to room temperature

  • Open the valve to reveal the inlet port

  • Pause the experiment on the relevant PromethION Flow Cell position(s) that will be refuelled:

    a. Navigate to Experiments and open the experiment running b. Click Pause c. A dialogue box will open. Choose the flow cell to pause and click Pause to confirm

PromethION

  • Set a P1000 pipette to 200 µl
  • Insert the tip into the inlet port
  • Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tip.
  • Using a P1000 pipette, load 250 µl of the FB into the inlet port of the flow cell, avoiding the introduction of air bubbles
  • Close the valve
  • Incubate the flow cell with the added FB for 3 mins
  • Unpause the experiment on the relevant PromethION Flow Cell position(s) after this process is complete by clicking Resume
  • (optional) Click Start pore scan and choose your flow cell, to pick a new set of the best channels for the remainder of the sequencing experiment Pore scan

The outcome and benefits of refuelling

Translocation speed and Qscore over time

Below is a graph that shows what is expected for translocation speed after the addition of FB to the flow cell in a previous version of MinKNOW.

As the speed drops below 300 bases per second, the Qscore begins to decline for the reads processed through the nanopores at this speed. After refuelling at the 17.5 hour mark, the speed begins to increase and returns to an improved rate (~400 bases per second), which is similar to the speed at the start of the experiment. After the addition of fuel, the quality of the data increases and returns to Qscores equivalent to those seen at the start of the run.

Refuel Arrow - 16 May 19

Refuelling multiple times in a run

You can refuel a sequencing run multiple times over an experiment. When you should refuel will depend on when the translocation speed drops below 300 bases per second on the speed graph in the MinKNOW GUI for Kit 9 (e.g. SQK-LSK109). Kit 14 (e.g. SQK-LSK114) adapter contains fuel fix technology, enabling users to run long experiments without the need for fuel addition.

CAUTION

Warning - overloading the flow cell when refuelling

If you refuel your flow cell multiple times, the waste reservoir of the flow cell will gradually fill up with buffer.

The array and waste reservoir of the below platforms (Flongle, MinION/GridION, PromethION) will take the following fluid loads:

  • priming mix
  • a library
  • one refuel - for Flongle
  • three refuels - for MinION/GridION
  • four refuels - for PromethION

42. Checks and monitoring

During the sequencing experiment, you can check various flow cell health and performance parameters that are shown in the MinKNOW GUI. Each parameter is described in more detail in this section.

Flow cell health

During a sequencing experiment, the MinKNOW Sequencing Overview page shows a flow cell icon with coloured bars. The bars represent the combined health of all pores in a flow cell, and indicate how well the flow cell is performing. The colours are:

  • Light green: sequencing
  • Dark green: open pore
  • Dark blue: pore recovering
  • Light blue: pore inactive

This information is identical to the last bar of the pore activity plot (described later).

a) Flongle/MinION/GridION flow cell health b) PromethION flow cell health

flow cell health images

Experiment summary information

The Experiments page displays summary information for all sequencing flow cells and device checks carried out on the device.

From this page, the user is able to control specific runs and identify real-time information including flow cell health and reads, giving users real-time feedback for sequencing flow cells.

  • Read count: The total number of reads across the experiment.
  • Estimated bases: The number of estimated bases to be basecalled from the experiment
  • Basecalled bases: The number of basecalled bases from the experiment.
  • Total run time: The duration of the experiment.
  • Temperature: The heatsink temperature of the selected position, which for sequencing should reach 34°C with Kit 9-11, 30°C with Kit 12 or 35°C on Flongle. For Kit 14, sequencing temperature is approximately 36°C.

experiment 2

TIP

As the MinKNOW script progresses, you can check the following:

  • Number of active pores
  • Heatsink temperature
  • Development of the read histogram
  • Pore occupancy
  • Local basecalling report

Channel states

The channel states pannel gives an overview of the states the flow cell pores are in to give the user an idea of how well the sequencing run is performing in real time. A good library will be indicated by a higher proportion of light green channels in "Sequencing" than are in "Pore available". The combination of "Sequencing" and "Pore available" indicates the number of active pores at any point in time. A low proportion of "Sequencing" channels will reduce the output of the run.

Channel states panel

Clicking on the Show Detailed button reveals a more detailed array of channel states:

  • Sequencing: Pore currently sequencing.
  • Pore Available: Pore available for sequencing.
  • Adapter: Pore currently sequencing adapter.
  • Active feedback: Channel ejecting analyte.
  • No pore: No pore detected in channel.
  • Multiple: Multiple pores detected. Unavailable for sequencing.
  • Unavailable: Pore unavailable for sequencing.
  • Unclassified: Pore status unknown.
  • Saturated: The channel has switched off due to current levels exceeding hardware limitations.
  • Out of range-high: Current is positive but unavailable for sequencing.
  • Out of range-low: Current is negative but unavailable for sequencing.
  • Zero: Pore currently unavailable for sequencings.

Pore activity plots

The pore activity plot summarises the channel states over time. Each bar shows the sum of all channel activity in a particular amount of time. This time bucket defaults to 1 minute, and scales to 5 minutes automatically after 48 minutes. However, bucket size can be adjusted in the "Bucket size" box in Display Settings.

The graph populates over time, and can be used as a way to assess the quality of your sequencing experiment, and make an early decision whether to continue with the experiment or to stop the run.

Pore activity GUI updated graph

To see the more detailed view of channel states, click Show detailed.

  • Sequencing: Pore currently sequencing.
  • Adapter: Pore currently sequencing adapter.
  • Pore available: Pore available for sequencing.
  • Unavailable: Pore unavailable for sequencing.
  • Active feedback: Channel ejecting analyte.
  • No pore: No pore detected in channel.
  • Out of range-high: Current is positive but unavailable for sequencing.
  • Out of range-low: Current is negative but unavailable for sequencing.
  • Multiple: Multiple pores detected. Unavailable for sequencing.
  • Saturated: The channel has switched off as current level exceed hardware limitations.
  • Zero: Pore currently unavailable for sequencing.
  • Channel disabled: Channel is disabled and awaiting another pore scan.

Read length histogram

The cumulative histogram shows reads compared to bases. Use the options below to choose the axis legends:

  • Y-axis: Estimated bases or basecalled bases
  • X-axis: Read length or read counts

Read count - this shows the number of reads vs read length. This enables you to understand how the read lengths vary in number and size.

Read length - this shows the total number of bases vs the read length.

The N50 value is presented (only for the whole set of passed reads) in the top left corner of the histogram.

26 Each histogram's X-axis (read length) can be zoomed in using the scaled bar under the histogram. Use Reset to refocus the zoom bar and histogram for the entire 'passed read' dataset. 29a You can see the number of bases by hovering over the bar in question.

Reads that are outliers in terms of length can be removed from the graph by ticking the Hide outliers box below the histogram.

Select Split by read end reason to view split reads and hover over for further information. This is useful for adaptive sampling which is further explained in the Adaptive Sampling info sheet.

  • Device changed MUX/Pore scan initiated: The strand ended because there was a scheduled pore scan that interrupted the read.
  • Read completed: The strand ended naturally as it passed through the pore.
  • Read blocked: The strand ended because it was deemed of low quality and purposefully rejected.
  • Adaptive sampling voltage reversal/Adaptive sampling rejection: The strand was rejected by adaptive sampling because it did not align to the target sequence when enriching, or the strand matched to the target sequence when depleting.

Read length histogram 1

Duplex output

This graph is available to display cumulative output of both simplex and duplex data, including unpaired and, optionally, paired simplex data. Data output can be displayed as bases or reads.Note, the complement and template split in the graph are based on estimates.

Note: This graph is only available when duplex basecalling is turned on and HD flow cells (R10.4.1HD) are used.

duplex output graph

Cumulative output

The cumulative output graph shows:

  • the number of bases that have been sequenced and basecalled

12

  • the number of reads that have been sequenced and basecalled; and whether the reads have passed of failed the quality filters

11

Cumulative output of multiple flow cells

If there are multiple flow cells running under the same experiment name, you will see the Experiment view. This gives output information on all assigned flow cells, plus a running cumulative total of bases or reads sequenced.

The cumulative output graph shows the running total number of Gbases sequenced by the GridION. image2022-7-29 12-15-37

The output generated by each flow cell to make the total cumulative output can be represented by individual output plots.

Both graphs can be switched between in the MinKNOW GUI. Reads, bases (estimated or basecalled) depending on the name, sample ID or flow cell ID can be selected to tailor the output graph, as required.

Temperature and Bias Voltage graph

Temperature vs time graph

The temperature graph gives a real-time representation of the heatsink temperature of the flow cell. If the temperature reading drifts out of the target zone, please consult Technical Services, otherwise the quality of your data may be compromised.

Please note, for our kits using Kit 14 chemistry, there will be some temperature fluctation at the beginning of the run due to speed normalisation. 18

Bias voltage vs time graph

The bias voltage graph provides the running voltage in real-time. MinKNOW will automatically adjust the applied voltage and will naturally drift to lower voltages as the electrochemistry in the flow cell is depleted. This graph is useful for running a flow cell multiple times.

If you set the voltage for a subsequent run as the final running voltage of the previous run, then MinKNOW will find it easier to identify the appropriate running voltage.

You will notice drops in the voltage at regular intervals and these will correspond to the channel scans that are defaulted to occur every one and a half hours. Here, each channel will be scanned to look for its availability for sequencing. The common voltage is reversed before and after each channel assessment for clearer results. 9

Translocation speed and quality score graphs

Note: These graphs are only present if Basecalling is turned on.

Translocation speed vs time

The translocation graph gives a real-time representation of the speed at which DNA/RNA strands pass through the pore. If the translocation speed drops below this window, data quality and output may be compromised as strands take longer to move through the pore.

Please note, translocation speed will be dependent on the kit chemistry used.

16

Quality score vs time

The Quality score graph gives a live representation of the median strand Q-score over time. Screenshot 2022-05-12 145130

Modal quality score graph is available across the run. This graph presents both the spread of quality scores and the modal quality score across the run, and the minimum quality score set. Screenshot 2022-05-12 145114

Q score histogram

This Q score graph will be generated over time. Both simplex and duplex Q score can be viewed and there is a zoom function available in the X-axis.

duplex q score

Barcode read counts

The Barcode Read Counts graph shows the breakdown of barcoded reads, if barcoding was used for the experiment. The default view only shows reads that have passed the quality score filters. However, selecting the Display failed box will show all reads. 18

The X-axis of the histogram also has a zoom function using the scaled bar underneath. Use Reset to refocus the zoom bar and graph.

Note that there is a small amount of cross-talk between barcodes. Some barcoded reads may appear on the graph even if the barcode in question was not used for the experiment.

Note: This graph is only present if barcoded is turned on.

Alignment hits

The alignment hits graph will populate when alignment and basecalling is set-up to run during sequencing. This bar graph shows the number of reads and bases that align to each of the entries in the user reference .fasta file or minimap index file. An entry in the reference file will only appear on the graph once a single read has aligned to it.

Screenshot 2023-07-18 at 12.00.37 PM

The X-axis of the histogram also has a zoom function using the scaled bar underneath. Use the Reset to refocus the zoom bar and graph.

A target alignment hits view is also available in the same graph by clicking Alignment coverage. This is populated by the .BED targets if a .BED file is supplied with the alignment reference.

Screenshot 2023-07-17 at 3.17.17 PM

Alignment Q accuracy histogram

The Alignment Q accuracy histogram will populate when alignment and basecalling is set-up to run during sequencing, displaying alignment accuracy. The graph is functional for both Duplex and Simplex runs.

Alignment Q accuracy histogram 5.9

Alignment and barcode heatmap

The alignment and barcode heatmap is only available when alignment and demultiplexing is performed with basecalling during sequencing. The heatmap graph shows the alignment hits split per barcode. The colour gradient shows bases and reads which are the more popular barcode and alignment hit combinations. Use the options below to view reads or bases and the slide bar above to focus on specific regions.

8

Note: These graphs are only present if barcoding and alignment are both turned on.

Traceviewer

The Traceviewer displays the current levels from individual channels. By default, it is set to show 10 channels. This number can be changed through the selection boxes beneath the viewer. Additional parameters that can be altered:

  • Time: The length of time plotted on one screen
  • Maximum: The highest current level to be shown on the y axis GridION traceviewer

Please note that viewing a high number of channels in the traceviewer may impact the speed at which the GUI is able to function.

Pore scan

As the sequencing protocol starts, a pore scan begins before the sequencing. There are four groups of active pores, and the pore scan allows MinKNOW to pick the best-performing pores in each group, maximising the data output in the initial stages of the run. The software also instantly switches to a new channel in the group if a channel is in the “Saturated” state, or after ~5 minutes if a channel is “Recovering”.

Pore scan GUI updated graph

END OF STEP

End of sequencing protocol script.

The length of the sequencing scripts are indicated during script selection, e.g. 72 Hr. However, if live basecalling is selected, a minimum-spec laptop may not keep up with the speed of data acquisition, and not all reads will be basecalled by the end of the experiment. The reads that had not finished being basecalled during the experiment will continue to be basecalled in Catch-Up mode.

The reads from the experiment will be found in the location set during experiment set-up in MinKNOW. The read file structure is described here. If the Desktop Agent is running simultaneously to MinKNOW, the full report will also be available. If it is being run at different times, the reads will be present in the data/reads folder ready to be processed.

43. Troubleshooting your run from the pore activity plots

Pore activity plot introduction

The pore activity plot feature in the MinKNOW software can be used to judge the quality of your experiment. The pore activity plot shows the distribution of channel states over time, grouped by time chunks, or 'buckets'. The basic view shows the five main channel states: Sequencing, Pore, Recovering, Inactive, and Unclassified. Clicking the "More" button shows a more detailed breakdown of channel states.

It is recommended to observe the pore activity plot populating over the first 30 min-1 hr of the sequencing run. By this time, the channel state distribution will give an indication whether the DNA/RNA library is of a good quality, and whether the flow cell is performing well.

If Active Channel Selection is enabled during the run, the software instantly switches to a new channel in the group if a channel is in the “Saturated” or “Multiple” state, or after ~5 minutes if a channel is “Recovering”. This feature maximises the number of channels sequencing at the start of the experiment, however this may also result in an artificially high number of "Sequencing" or "Pore" channels in the pore activity plot. For this reason, we recommend referring to the pore scan plot, which shows the true distribution of channel states at the point of the most recent pore scan.

Good library

A good quality library will result in most of the pores being in the "Sequencing" state, and very few in "Pore", "Recovering" or "Inactive". A library that looks like this is likely to give a good sequencing output.

image2022-3-25 14-45-0

Channel blocking

Under certain conditions (usually the presence of contaminants in the library), pores may become blocked and therefore unable to sequence. This manifests itself as a build-up of "Unavailable" pores over time.

image2022-3-25 10-43-25

Recommendation:

  • If, despite the channel blocking, the library is still producing a sufficient number reads to answer your biological question, you can carry on with the sequencing experiment.
  • Otherwise, stop the sequencing run in MinKNOW. Then wash out the library from the flow cell using the instructions for the Flow Cell Wash Kit. Then prepare another library and load it on the flow cell.

Osmotic imbalance

If the plot shows a high number of "Inactive" channels building up over time, this could indicate that the channels or membranes have been damaged, for example by air bubbles, osmotic imbalance, or the presence of detergents or surfactants in the library.

Pore activity: image2022-3-25 14-46-40

Pore scan: image (29)

Recommendation:

  • Check the channel panel: if the Inactive channels are all grouped in one part of the flow cell, this could indicate an air bubble that has been introduced during flow cell flushing or library loading. If the remaining channels are still sequencing, it is possible to carry on with the run. Do not try to move the air bubble, as this can damage even more channels.

image2022-3-24 19-9-17

If the Inactive channels are distributed throughout the flow cell:

  • Check that the heat tape on the underside of the flow cell is intact.
  • Make sure that the input DNA is in either TE buffer or nuclease-free water, and that the buffer contains no detergents or surfactants.
  • Make a new batch of flow cell priming buffer (a mixture of Flush Buffer/Flow Cell Flush and Flush Tether/Flow Cell Tether). Flush the flow cell with the mixture and load the library again.

Low pore occupancy

If there was insufficient starting material, or some sample has been lost during library prep, or the sequencing adapters did not ligate well to the strand ends, the plot will show a high ratio of "Pore available" to "Sequencing" states, meaning that only a limited number of pores are sequencing at any one time.

image-20230202-093953

Recommendation:

  • Check the amount of DNA/RNA in your prepared library, for example by using the Qubit fluorometer. We recommend preparing a fresh library and reloading your flow cell with the recommended loading input from the relevant protocol
  • If your library is at a low concentration, prepare the library again using a higher amount of starting material.
  • Ensure you are adding the flush tether to your priming mix during flow cell priming and loading before adding your library.
  • For ligation-based protocols, ensure you are following the protocol to ligate the sequencing adapter correctly and performing any clean-up steps after adapter ligation with short or long fragment buffer rather than ethanol.

RNA pore activity

For RNA in particular, the expanded channels view may show a large proportion of pores sequencing Adapter. This happens because RNA strands are usually shorter than DNA, and the adapter takes up a larger proportion of the strand. Additionally, the RNA sequencing chemistry is optimised for sequencing RNA, whereas the adapter is DNA, and is processed slower. As long as the 'basic' pore activity plot view shows the majority of pores in "Sequencing", a high proportion of Adapter should not be a problem.

image2022-3-24 19-17-12

44. Completion of a sequencing protocol script

Catch-up notifications

Catch-up basecalling is carried out either after the sequencing run completes (e.g. after 72 hours), or if the run is manually terminated. To manually terminate a run, select sequencing flow cells and click Stop.

step8

A pop-up window will appear: stop1

A progress bar is displayed during catch-up basecalling on the Sequencing Overview and Experiments pages.

IMPORTANT

Please note that in the current version of MinKNOW, stopping your experiment and stopping basecalling through the GUI when ONLY FASTQ output is selected leads to queued reads being discarded. We are working on a fix.

CSV files output by MinKNOW

The data used by MinKNOW to generate the pore activity and Cumulative Output plots are output as CSV files. These files are in the run-specific folder in the reports directory (e.g. \data\reports on Windows).

Using the data in the CSV files, users can recreate pore activity and Cumulative Output graphs themselves in Excel by adding the various pore states to the 'Legend Entries (series)' panel:

CSV file

Below are definitions of the columns in the output CSV file from MinKNOW:

Column title Description
Experiment time (minutes) Time in minutes from the beginning of the sequencing run.
Reads Cumulative number of reads basecalled since the beginning of the sequencing run.
Basecalled reads passed Cumulative number of reads basecalled since the beginning of the sequencing run with a Qscore above the filter threshold.
Basecalled reads failed Cumulative number of reads basecalled since the beginning of the sequencing run with a Qscore below the filter threshold.
Basecalled reads skipped Cumulative number of reads that have not been basecalled since the beginning of the sequencing run.
Selected raw samples Raw analog-to-digital converter (ADC) samples that have been identified by MinKNOW as part of a read.
Selected events Measure MinKNOW uses to estimate bases and distinct from events used in basecalling.
Estimated bases Number of (MinKNOW) events multiplied by some fixed factor defined per chemistry.
Basecalled bases Number of estimated bases that have been in all selected reads above.
Basecalled samples Number of basecalled bases that have been in all selected reads above.

Acquisition termination file for LIMS

Customers using LIMS will receive a summary file once data acquisition and file writing has completed. The file is named final_summary.txt, and is written out to the output directory.

An example of the file contents is shown below:

instrument=PCT0009
position=1-A5-D5
flow_cell_id=PAD15520
sample_id=Chinese_NA24631
protocol_group_id=JB_47x_P48005_270319
protocol=4a37d56a62b6425a2af4d01d4970c7b5cc7b7c80
protocol_run_id=2a4ccaa1-8aaf-4872-a1f3-773a686d64de
acquisition_run_id=7eeaea330d1fe2ec43583072bbe21288254bef1b
started=2019-03-27T17:23:55.542284+00:00
acquisition_stopped=2019-03-28T09:58:37.505691+00:00
processing_stopped=2019-03-28T09:58:37.505788+00:00
basecalling_enabled=1
sequencing_summary_file=sequencing_summary/PCT0009_20190327_172337_PAD15520_promethion_sequencing_run_Chinese_NA24631_sequencing_summary.txt
fast5_files_in_final_dest=1606
fast5_files_in_fallback=0
fastq_files_in_final_dest=1197
fastq_files_in_fallback=0

45. Run report

Run reports overview

Run reports contain information about the sequencing run and include performance graphs. These graphs are automatically generated at the end of a run but can be manually generated by clicking Export run report and selecting which experiment to export to html.

The run report includes the run summary and configuration, sequence output, run health, and run log. This html report is interactive, allowing users to jump to the information they need and hide other details they do not require. Troubleshooting suggestions are also available throughout the report, with links to further information available on the Community.

Run summary

This is an overview of the sequencing run, including output and baseacalling results along with the duration of the run.

Screenshot 2023-07-10 142758

During a run: When a run report has been generated during a run, the elapsed time will show in the progress bar, along with:

  • The estimated time until flow cell end of life (if selected)
  • The estimated time until the data target condition will be met and if the flow cell will reach the target during the run time or flow cell life (if selected). A warning will appear under the progress bar if the data target will not be met.

If a run report was generated when a sequencing run is paused, the run status will only display "PAUSED" with no progress bar or estimations.

Run report paused

After a run: When a run report is generated after a sequencing run has completed, the duration of the run will be displayed, along with:

  • The run status confirming the flow cell end of life condition was met and completed progress bar.
  • The run status confirming the data target was met and a partially filled progress bar reaching the data target. If the data target was not met, a warning will appear under the completed progress bar.

Note: The relevant run until parameters must be defined during the sequencing run set up for these status reports to appear on the run report.

Run configuration

A detailed overview of the settings selected for the sequencing run and data output, including the software versions used to sequence the data. We have also included further information about the modified bases and basecalling models chosen, barcode balancing settings, expansion kits used and read splitting preferences selected.

Run report run configuration

Sequence output

This section includes a more detailed view of the sequencing output, such as read lengths, barcodes detected and quality score.

Read lengths The total number of bases against the read length are displayed in this graph. Any outliers displayed in a table below, displaying read length and aggregated reads. Run report seq output read lengths

Barcodes Barcodes detected are displayed in a searchable table which can be exported as a .CSV file. Two graphs for bases and reads are available to view and displays total number of reads/bases above a Qscore of 9. Passed and failed data is also displayed and can be sorted by barcode.

Detected barcodes: Updated Run report barcodes detected

Bases graph: Updated Run report bases graph

Reads graph: Updated Run report reads graph

Cumulative output Total output of the bases and reads sequenced during the experiment are outlined in the graphs here. run report cumulative output

Quality score Quality score (Qscore) of your sequencing run is displayed here, with the minimum value set on MinKNOW during run set up. Run report seq output quality score

Alignment If an alignment file was supplied and alignment turned on during run set-up, the Reference alignment and Bed regions sections will be displayed with tables that can be exported as CSV files.

Reference alignment: This table will display the specified reference sequence and the number of reads, bases and coverage percentage.

Bed region: This table will display the specified reference sequence with the coordinates of the specific target within the reference sequence as well as the number of reads, bases and coverage percentage.

run report alignment references 2

Run health

This provides a detailed view of the pore activity and pore scan results throughout the run, along with graphs displaying the translocation speed and temperature.

Pore activity states:

  • No pore from scan: No pore detected in channel
  • Sequencing: Pore currently sequencing
  • Adapter: Pore currently sequencing adapter
  • Pore available: Pore available for sequencing
  • Unavailable: Pore unavailable for sequencing
  • Active feedback: Channel ejecting analyte
  • Out of range-high: Current is positive but unavailable for sequencing
  • Multiple: Multiple pores detected. Unavailable for sequencing
  • Saturated: The channel has switched off as current levels exceed hardware limitations
  • Out of range-low: Current is negative but unavailable for sequencing
  • Zero: Pore currently unavailable for sequencing
  • Channel disabled: Channel is disabled and awaiting another pore scan

Pore activity updated graph

Pore scan states:

  • Pore available: Pore in channel available for sequencing
  • Reserved pore: Pore in reserve, will return to available when required
  • Unavailable: Pore inhibited from sequencing
  • Saturated: Possible contamination in the sample
  • Zero: No current is passing through this pore, possibly due to bubbles on the membrane
  • Inactive: Pore no longer suitable for sequencing

Pore scan updated graph

Run report run health translocation temperature

Run log

The run log includes system messages sent during the sequencing run regarding errors, warning, and any events.

Run log system messages

46. Closing MinKNOW and shutting down a MinION Mk1B/Mk1D

Ensure that the flow cell is not running an experiment.

no running exp

Close MinKNOW by closing down the GUI.

Disconnect the MinION Mk1B/Mk1D.

47. Closing MinKNOW and shutting down on GridION

Shutting down your GridION

The GridION's computer requires a stepwise, processed shut down, otherwise you may face problems e.g. when recognising flow cells.

Follow the instructions in this section to ensure you do not face errors with your GridION from incorrectly shutting down your device.

Ensure none of the flow cells are running an experiment.

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

Navigate to Device Settings and click 'Shutdown'.

shut down

Turn off the device at the mains supply, if you are using mains power.

If connecting to a device remotely using a computer not directly connected to the device, close down the MinKNOW application window.

48. Closing MinKNOW and shutting down on PromethION

Shutting down your PromethION

The PromethION's computer requires a stepwise, processed shutdown, otherwise you may face problems e.g. when recognising flow cells.

Follow the instruction in this section to ensure you do not face errors with your PromethION from incorrectly shutting down your device.

Ensure none of the flow cells are running an experiment.

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

no runs prom

Navigate to Device Settings and click 'Shutdown'.

shut down

IMPORTANT

Do NOT select 'Reboot'. This will lead to errors in recognising the flow cells when commencing an experiment.

Once the screen goes blank, turn off the sequencing module and tower via the switch at the rear of the device.

If connecting to a device remotely using a computer not directly connected to the device, close down the MinKNOW application window.

49. Post-run basecalling

Basecalling overview

A user can basecall and demultiplex their data directly in MinKNOW after a sequencing experiment has finished to avoid having to use command-line tools, or to re-analyse old data using the latest basecalling models. Reads can also be aligned against a reference post-sequencing.

Note: Both barcoding and alignment can be run on FASTQ, POD5 or FAST5 reads when coupled with basecalling.

Click 'Analysis' on the start page.

analysis 1

Click 'Basecalling' to open the post-run basecalling set-up options.

basecalling

Select input folder containing the files to basecall from a previous run.

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.

Post run basecalling 1

Select output folder and file type.

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.

Post run basecalling 2

Choose basecalling model from the drop-down menu.

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.

Post run basecalling 3

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

OPTIONAL ACTION

Select any barcoding kits used for the run from the drop-down menu.

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).

Post run basecalling 4

OPTIONAL ACTION

Select to use alignment and import a reference sequence in a FASTA file.

We currently only recommend uploading an alignment reference locally for bacterial-sized genomes. Upload can take a few minutes and is compute dependent.

A reference file must by uploaded as a FASTA file that can contain multiple entries in the same file (e.g. multiple chromosomes).

However, a .bed file may also be uploaded alongside the reference FASTA file. The BED file option can be used when the user is interested in a particular region of the reference (e.g. specific gene in the chromosome). Alignment generates FASTQ output files when performed with basecalling.

Post run basecalling 5

Click 'Start' to begin post-run basecalling.

Post run basecalling 6

50. Post-run barcoding

Barcoding overview

A user can barcode their data directly in MinKNOW after a sequencing experiment has finished, using FASTQ data.

Click 'Barcoding' in the analysis menu to open the post-run barcoding set-up options.

Barcoding

Select the input folder containing the FASTQ data to barcode from a previous run.

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.

Post run barcoding 0

Choose output folder for the post-run barcoded data.

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.

Post run barcoding 1

Select any barcoding kits used for the run from the drop down menu.

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).

Post run barcoding 2

Click 'Start' to begin post-run barcoding.

Post run barcoding 3

51. Post-run alignment

Alignment overview

A user can align their data directly in MinKNOW after a sequencing experiment has finished, using a FASTQ file from a previous run.

The reference file of bacterial-sized genomes must be uploaded locally as a FASTA file. Alignment hits from these files are used to populate the alignment graphs.

A BED file may also be uploaded alongside the reference FASTA file when the user is interested in a particular region of the reference (e.g. specific gene in the chromosome). The BED file alignment hits will be highlighted in the sequencing .txt file generated in the data folder.

When alignment is run independently from basecalling, BAM files are generated.

Click 'Analysis' on the start page.

analysis 1

Click 'Alignment' to open the post-run analysis set-up options.

Alignment1

Select the input file containing the FASTQ data to carry out alignment from a previous run.

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.

Post run alignment 1

Choose the output folder for the post-run aligned data.

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.

Post run alignment 2

Select the alignment reference to carry out the post-run alignment.

The alignment reference must either be a FASTA file.

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

Post run alignment 3

Click 'Start' to begin post-run alignment.

Post run alignment 4

Last updated: 11/22/2024

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