Troubleshooting a NextSeq 500/550 An existing connection was forcibly closed by a remote host error

Important note: A mitigation for this error is available with NOS v1.7 and NCS v4.2. Upgrading Instrument's Control Software version is highly recommended.

Error message text:

• An error occurred while sequencing. Will now proceed to determining whether to perform a post-run wash. (System.Net.Sockets.SocketException)

• An existing connection was forcibly closed by the remote hostError code is "10054".

• Unable to read data from the transport connection: An existing connection was forcibly closed by the remote host.

Usually, this error will stop the run at cycle 5, as this is when Real Time Analysis (RTA) begins primary analysis for a run. The error usually happens when communication between Single-Board Computer (SBC) and Main Board (MPCA) boards are blocked (or the connection between the two is intermittent, or the MPCA is no longer working properly). A gateway IP for this internal connection can confuse it. In some circumstances, the internal network may have a gateway value, yet the NextSeq Control Software (NCS) was able to initialize fine. Troubleshooting steps:* Check to see if the output folder is set to save locally instead of to an external location, such as a network.

• Check which other processes or applications are running concurrently at the time of the error in Task Manager.

• Check if any gateway values have been changed (either manually or through a GPO). Note that Network Interface Cards (NIC) configurations must never be changed and IT must not push NIC changes remotely.

  • For more information, search for Knowledge Base article Troubleshooting NVRAM or Socket Errors on NextSeq 500/550

• Collect the group policy object (GPO) report and check the GPO report to make sure that no updates are being pushed.

• Check the Antivirus (to make sure that the antivirus software is not causing updates).

If this is the first instance of the error and no issue is found in the items above, power cycle the instrument and monitor the next run.* For more information, search for Knowledge Base article How to Perform a NextSeq 500/550 Power Cycle.

If another “An existing connection was forcibly closed by a remote host" error occurs after starting a new run, contact Illumina Technical Support for further assistance and request an FTP link to provide the files below.

Files to collect:* NextSeq Control Software (NCS) logs

• Windows 7: C:\Illumina\NextSeq Control Software\Logs

• Windows 10: D:\Illumina\NextSeq Control Software\Logs

• Universal Copy Service (UCS) logs: C:\ProgramData*\Illumina\UniversalCopyService

• RTA logs: D:\Illumina\ NextSeq Control Software Temp<Run_Folder>\RTALogs

• GPO report *Note: The ProgramData folder may be hidden; to un-hide files, select the View tab in the file explorer window and make sure Hidden items is checked.

For any feedback or questions regarding this article (Illumina Knowledge Article #2361), contact Illumina Technical Support [e-mail].