The PromethION 2 (P2i) is a benchtop device that runs up to two PromethION Flow Cells. Each flow cell is independently addressable, meaning that you can run experiments concurrently or individually. The P2i is a standalone product that includes enterprise-grade compute that performs all data acquisition and basecalling on the device.

The device has an integrated screen for interacting with the sequencing software, MinKNOW. This allows you to start, stop, and monitor sequencing runs as well as access other analysis features in MinKNOW. The device also has connectivity on the rear to plug in an external display, which enables access to the Ubuntu operating system/desktop and to the analysis software, EPI2ME.

*Functional range of electronics +5ºC to +40ºC

• 1x PromethION 2 Integrated
• 5x C13 power cables (1x US, 1x UK, 1x EU, 1x CN, 1x AUS) for international use
• 2x Configuration Test Cells (CTCs)

Status dots There are two coloured dots on the integrated display of the device to indicate the flow cell status: A and B, one for each flow cell position. They display the following patterns:

• Status dots are solid white: Device ready, no flow cells inserted.
• Status dots are solid blue: Flow cells inserted.
• Status dots pulse green: Script is running/data acquisition in progress (this includes the hardware check and flow cell check).
• Status dots solid red: Run finished with error. Contact Support if you see this pattern.
• Status dots are solid blue: Script ended successfully.

• Place the device on a strong, clean laboratory bench.
• Allow at least 30 cm clearance all around the device to ensure the sequencing temperature is stable throughout your sequencing run, resulting in the highest quality data.
• Do not cover any ventilation grilles.
• Carry out sequencing at a room temperature of +18°C to +22°C.
• Ensure that the Ethernet (RJ45) and mains power sockets are in close proximity.
• Ensure that the power socket is easily accessible should you need to disconnect the device in case of an emergency.

Before you install the device, make sure you have the following:

• 1x Ethernet cable (cat 5e or greater for max 2.5 Gbps speeds)
• Network access providing:
  • IP address through DHCP
  • Outbound-only access to the internet over port 443
  • Firewall permissions for access to AWS eu-west-1 IP ranges listed here, in addition to: 178.79.175.200 and 96.126.99.215

Note: If you are using an optional external display for accessing the Ubuntu desktop or EPI2ME, plug in the monitor, keyboard, and mouse, and power on the monitor before switching on the P2i. If you only use the integrated screen, do not plug in the keyboard and mouse as this will cause the device to boot incorrectly.

• 1x monitor (HDMI/DisplayPort compatible)
• 1x appropriate monitor cable
• 1x USB connected keyboard
• 1x USB connected mouse

Optional: Uninterruptable power supply (UPS) to provide temporary power during an outage. The P2i has not been validated against any model of UPS.

For first time installation, we recommend using an external monitor (HDMI/DisplayPort compatible), keyboard, and mouse. To be recognised correctly, connect the peripherals before switching on the device. Continued use of the device does not require the monitor, keyboard and mouse, and they can be unplugged if not required.

All Oxford Nanopore devices use MinKNOW™ as the primary software. The MinKNOW software carries out several core tasks: data acquisition, real-time analysis and feedback, basecalling, data streaming, controlling the device, and ensuring that the platform chemistry is performing correctly to run the samples. MinKNOW takes the raw data and converts it into reads by recognition of the distinctive change in current that occurs when a DNA strand enters and leaves the pore. MinKNOW then basecalls the reads, and writes out the data into POD5, FAST5 or FASTQ files.

Please update MinKNOW to the latest version when setting up your P2i device for the first time using the instructions in Updating the software via the UI.

The P2i supports a maximum transfer speed of 2.5 Gbps using the rear Ethernet port. This connection is used to connect to the local infrastructure. The P2i does not support Wi-Fi.

The Ethernet connection will be used for:

• Sending telemetry* information to Oxford Nanopore Technologies
• Receiving software and firmware updates to the device
• Streaming biological sequence data to your local storage infrastructure

To connect the P2i into the local infrastructure, you need to provide all the appropriate cables.

*Telemetry contains metadata about the settings and conditions of a sequencing run. Nothing specific about the genomic content of individual reads is included - only generic information is logged, such as sequence length and q-score.

If you want to connect your P2i to a single network, you can use the MinKNOW user interface.

Note: You will need to use an external monitor to configure the network via the Terminal. Plug in and power on the monitor before switching on the P2i. If you plug in the monitor while the P2i is powered on, the device will not recognise the monitor.

To configure your P2i to connect to multiple networks, open a Terminal window and enter:

/opt/ont/mooneye/bin/ont-mooneye-configure-lan

To see help options, run:

sudo /opt/ont/mooneye/bin/ont-mooneye-configure-lan -h

You can use the command:

sudo /opt/ont/mooneye/bin/ont-mooneye-configure-lan -v -s

to validate that the IP address has been set correctly using a static or DHCP address.

Note: You will need to use an external monitor to update the hostname via the Terminal. Plug in and power on the monitor before switching on the P2i. If you plug in the monitor while the P2i is powered on, the device will not recognise the monitor.

If you need to update the hostname of the device, run the following command and replace the <new_hostname> portion with your desired hostname (omitting the angle bracket characters <>):

sudo -S hostnamectl set-hostname <new_hostname>

Power cycle the device by shutting down with the following command, then press the physical power button to turn the device back on:

sudo shutdown -h now

To set the the NTP server to e.g. "ntp.ubuntu.com", enter the following command:

sudo /opt/ont/mooneye/bin/ont-mooneye-time-source -a -v --ntp-server=ntp.ubuntu.com

To verify that the time server is set:

sudo /opt/ont/mooneye/bin/ont-mooneye-time-source -v -s

For more detailed sync information, enter:

sudo timedatectl timesync-status

A loading bar will be displayed under the CTC during the checks.

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 three months of purchase, provided that you report the results within two days of performing the flow cell check and you have followed the storage recommendations.

The quality of the flow cell will be shown as one of the three outcomes:

1. Yellow exclamation mark (Flongle flow cell): The number of sequencing pores is below warranty. Take the flow cell out of the device, re-insert it and run a flow cell check again. If the flow cell is still below warranty, contact support@nanoporetech.com

2. Green tick (MinION flow cell): The number of sequencing pores is above warranty.

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

Note: The indicator of quality will only remain visible during a MinKNOW session. Once the MinKNOW session has ended, the status of the flow cell will be erased.

To run a sequencing experiment, you will need your DNA or RNA sample purified and a sequencing library prepared. For more information about sample preparation, refer to the Prepare section of our Documentation.

You can access the MinKNOW protocol by following this link.

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.

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

If your P2i runs out of storage during a sequencing run, your experiment will prematurely terminate. Therefore, before beginning an experiment, ensure that you have at least 2 TB of storage per flow cell.

For data transfer and long-term storage, we recommend using a remote server or network-attached storage (NAS). These typically offer a choice of NFS, SMB, or CIFS shares. We recommend using NFS mounts due to the supported Linux user and group permissions. For Windows workstations, use SMB. For Mac OS use either SMB or NFS. To stream data to storage in real-time, a SSD is required due to its high write speed compared to HDD. After initial writing to networked SSD drives, you can move the data to storage with a slower write speed for long-term storage.

The form and volume of data to be stored will depend on your requirements and whether you wish to basecall your sequencing data in the future when more advanced basecalling algorithms are available:

Storing .pod5 or .fast5 with raw read data in will permit re-basecalling of data when new algorithms are released by Oxford Nanopore Technologies. In such cases, new releases of basecallers have enabled significant improvements in basecalling accuracy of existing datasets through re-basecalling. Further, selected Oxford Nanopore and third-party tools use the raw signal information contained within the .fast5 to extract additional information from the raw signal, e.g. calling modified bases, reference-guided SNP calling, or polishing of data.

Retaining only FASTQ files will allow use of standard downstream analysis tools using the DNA/RNA sequence, but no further sequence data can be generated when improvements in basecalling become available.

Oxford Nanopore is unable to provide exact recommendations for storage, as these will be site-specific.

Our device FAQs are located here.

• 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.

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.

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