Ion PGM™ Sequencing 400 Kit - FAQs

You may, but W2 may run out in the middle of the second run. However, you can collect the data right before W2 runs out and reanalyze it after the run finishes. If you want complete data, please perform only one run per initialization.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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.br/>
Please note that passing pH values for the reagents may vary depending on the sequencing kit used, but the instrument will automatically determine the correct pH range depending on the sequencing kit selected during initialization. If you are performing the reagent check outside of the initialization, please contact Technical Support for guidance.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Each Ion PGM Sequencing kit includes reagents and materials for four initializations. The number of runs per initialization will depend on the desired read length.

The Ion PGM Sequencing 200 Kit v2 (Cat. No. 4482006) supports two runs per initialization (or eight runs per kit) for 200–base read sequencing. For ≤ 100–base read sequencing, there are enough reagents to support three runs per initialization (or twelve runs per kit).

The Ion PGM Sequencing 400 Kit (Cat. No. 4482002) supports one run per initialization (or four runs per kit for 300– and 400–base read sequencing).

Please see the “Product Information” section of the Ion PGM Sequencing kit user guides for additional information.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Currently, the Ion PGM System supports up to 400–base reads.

For up to 400-base reads, using the Ion PGM Hi-Q View Chemistry and Ion PGM One Touch 2 system, we recommend:
• Ion PGM Hi-Q View One Touch 2 Kit (Cat. No. A29900)
• Ion PGM Hi-Q View Sequencing Kit (Cat. No. A30044)

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Signal Processing Input - Required to reanalyze an Ion Torrent Ion PGM or Ion Torrent Ion Proton run from scratch. (The Output file category is also required).

Basecalling Input - Required to re-analyze an Ion PGM or Ion Proton run from basecalling. (The Output file category is also required).

Output - Required to see the run report in the Torrent Browser and either to reanalyze the run or to launch a plugin.

Intermediate - Useful to troubleshoot a run. Not required for the run report or for re-analysis.

Intermediate files can be deleted if not needed for run troubleshooting. Depending on your institution, you may need to keep all other files (Signal Processing, Basecalling, and Output).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Follow these steps to change the DNA barcode ID:

- Open a run reports listing in the “Data,” “Completed Runs & Reports” tab.
- In the list view, click the “Edit” button on the right side of the run listing. The Edit Run window opens.
- Click the barcode menu pull-down and choose the correct barcode set. If you have custom barcode sets, your list is different. Click the “Save” button (in the bottom right corner).
- You can now reanalyze the run from the “Data,” “Completed Runs & Reports” tab.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Log in to Torrent Browser and click the “Data” tab, “Completed Runs & Reports page”, and find your run name. In the table view, click the “Reanalyze” option in the gear menu on the right of the run entry. The main run analysis dialog opens. Click the “Advanced” button to display the additional run parameter options. Click the “Alignment Reference” pull-down menu and select the reference for this run. (Optional) Change other advanced options if required. (See “Work with Completed Runs” for a description of the advanced fields.) Click the “Start Analysis” button.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

The GGCCAAGGCG sequence is part of the RNA library adapter sequence. This sequence can be automatically trimmed by the software by selecting the “RNA_Barcode_None” option under the Barcodes menu. The run can be reanalyzed with the correct barcode settings to trim the adapter sequence. In the future, the “RNA_Barcode_None” option should be selected in the run plan or selected on the instrument prior to starting the sequencing run.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Please refer to the Data Management section of the Torrent Suite Software user guide for your version of software. The user guides can be found here under Documents: https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/next-generation-sequencing-support-center/ngs-software-support.html

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Primary analysis and secondary analysis of the data is performed by the Ion Torrent Server. Depending on the application, there may be tertiary analysis plugins that can be installed on the Torrent Server. For other applications, such as RNA-Seq, we recommend using third-party software. For more information, please see the Ion NGS Analysis Workflow page (https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-data-analysis-workflow.html).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

You can find user guides, release notes, and additional information on the Torrent Suite Software here (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/next-generation-sequencing-support-center/ngs-software-support.html).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

For more information on the informatics workflow and analysis software, such as Ion Reporter and Torrent Suite, please go to the Ion NGS Informatics workflow page (https://www.thermofisher.com/us/en/home/life-science/sequencing/next-generation-sequencing/ion-torrent-next-generation-sequencing-workflow/ion-torrent-next-generation-sequencing-data-analysis-workflow.html).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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, which will take 3-4 hours. If you have a monitor and keyboard connected directly to the Torren Server, you can avoid the system check 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.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

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.

Please note that passing pH values for the reagents may vary depending on the sequencing kit used, but the instrument will automatically determine the correct pH range depending on the sequencing kit selected during initialization. If you are performing the reagent check outside of the initialization, please contact Technical Support for guidance.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Please refer to the “How to assess a PGM sequencing run report” in the Documents section of the NGS Sequencing Support Center (https://www.thermofisher.com/us/en/home/technical-resources/technical-reference-library/next-generation-sequencing-support-center/ngs-support.html).

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Firmware updates for Ion instruments are included with the latest version of Torrent Suite Software. First, download and install the latest version of Torrent Suite Software on your Torrent Server. Once completed, your Ion PGM System should alert you that an update is available. You can also start the update process from the Options menu. From the Main menu, select Options, then Updates, Released Updates, and Check. Once your PGM system has detected the update, press the Update button to begin the process. Instructions to update the Ion PGM Sequencer can also be found in the Appendix of the Ion PGM sequencing kit user guides.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

The Ion Hi-Q View Sequencing Kit (Cat. No. A30044) includes sequencing supplies, sequencing solutions, and sequencing dNTPs for eight 200 bp sequencing runs or four 400 bp sequencing runs.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.

Please see the Ion PGM System Product Selection Guide (http://tools.thermofisher.com/content/sfs/brochures/Consumables-Product-Selection-Guide.pdf) for a list of available products for the Ion PGM System workflow organized by application.

Find additional tips, troubleshooting help, and resources within our Next-Generation Sequencing Support Center.