NGS Sequencing Support-Troubleshooting

The pH of the nucleotides may be out of range or there may have been a minor problem during measurement. Press the “Start” button and restart the measurement. If it passes, continue to start the run. If an error appears again, please note the pH of R1, R2, R3, and R4, as well as the error message, and contact Technical Support.

The pH of W1 may be out of range, or the volume of W1 may be too small, or there may have been a minor problem during measurement. Check the amount of W1, press the ”Start” button on the screen, and restart the measurement. If it passes, continue to start the run. If an error appears again, please note the pH of W1, the amount of W1, as well as the error message, and contact Technical Support.

The Ion PGM™ System and Torrent Server may not be connected. Please shut down the system and server and reboot them. The server may enter a system check after the reboot. Since the system check will take 3–4 hours, you can avoid it by pressing “c” on the keyboard.

If there is a reason you cannot reboot the system, you can run the Ion PGM™ System without connection to the server. The runs will be saved inside the system. For 200 bp sequencing, here is the number of runs that may be saved for these chip types:

• Ion Torrent™ Ion 314™ chip: 40 runs
• Ion Torrent™ Ion 316™ chip: 6 runs
• Ion Torrent™ Ion 318™ chip: 5 runs

The data will automatically transfer to the server after connection.

Please check the following:

1) That there is solution left in the W1 bottle

2) That the sippers and bottles are not loose

If the sippers and bottles are not loose and there is at least 200 mL of solution in the W1 bottle, the line between W1 and W2 may be blocked. Please run the line clear.

If the same error message appears even after the line is clear, detach the reagent bottles from the Ion PGM™ System, water-clean, and shut down the instrument. Cap the reagent bottles. Restart the instrument, and check the Ion PGM™ chip. Open the chip clamp and check that there is no solution standing in the port of the chip. If there is some solution, change the chip to another used chip or a new chip.

Place the W2 and W3 bottles back on the Ion PGM™ System. Discard the solution inside the W1 bottle, make more solution by adding 350 µL of 100 mM NaOH, and place it back on the Ion PGM™ System. Restart initialization.

Open the chip clamp and check for any leaks. If you see solution leaking, please remove the chip and clear the solution with a Kimwipe™ tissue. DO NOT wipe or scrub, as the socket could be damaged. Clean the socket by gently touching it with a damp Kimwipe tissue. Make sure the socket is dry and replace the chip. Press the “Calibration” button to recalibrate the chip. If it fails again, change the chip to a different one. Also, if you are using an Ion 316™ chip v2 on a v3.4 or older instrument, the instrument will not recognize the chip.

Rinse the Proton chip by pipetting 100 μL of isopropanol into the chip, followed by 100 μL of water. This may remove any bubbles or reside from the chip surface that may cause initialization issues.

This may indicate a fan failure inside the instrument. Please call 1-87-SEQUENCE to open a service call.

After inserting a new chip and prior to pressing the “Next” button, please wait until a green check mark is displayed on the bottom left of the instrument screen. This indicates that the chip is detected. 

Here are possible causes and recommended actions to take:

Possible causes:

• Software updates available 
• Connectivity issues
• Instrument not detecting required files or hardware

Recommended actions to take (click on the red pop-up to see detailed messages);

• If a message states "Newer Software Available": 

IMPORTANT! After updates are installed, the instrument must be restarted. 

a. In the Main Menu, select Options>Updates. 

b. Select the Released Updates checkbox, then press Update. 

c. When installation is complete, follow the onscreen prompts to restart the instrument. 

Note: In some cases, the instrument restarts automatically after software installation. 

• If a message states "No Connectivity to Torrent Server", "No Connectivity to ftp server", or "Network Manager not connected", disconnect and re-connect the ethernet cable, confirm that the router is operational, and verify that the network is up and running. 

• For any other messages: 

a. Power off the instrument: In the Main Menu, select Tools > Shut Down > Shut Down. 

b. Wait 30 seconds, then press the button on the front of instrument to power on the instrument. 

• If the red "Alarms" and/or "Events" message still appears in the main menu, contact Technical Support.

This could be either due to poor chip loading or because Control Ion Sphere™ particles were not added to the sample. Please confirm that the Control Ion Sphere™ particles (included in the Ion S5™ Installation Kit) were added. If controls were indeed added, contact Technical Support for assistance.

This indicates a problem with library or template preparation. We recommend verifying the quantity and quality of the library and template preparations.