Flow Cell Check

Version for device:

Other available device versions: MinION Flongle

1. Purpose of the flow cell check

   A Flow Cell Check is used to assess the number of nanopores that are available in the flow cell. It should be run when you are ready to use the flow cell, and within 3 months of purchase (except for Flongle flow cells, which currently have a shelf life of four weeks).

   Oxford Nanopore Technologies will replace any flow cell that falls below the warranty number of active pores, provided the result is reported within two days of performing the Flow Cell Check and the storage recommendations have been followed.

   Flow cell	Minimum number of active pores covered by warranty
   Flongle Flow Cell (FLO-FLG001)	50
   Flongle Flow Cell (FLO-FLGOP1)	30
   MinION/GridION Flow Cell	800
   PromethION Flow Cell	5000

2. Flow cell storage instructions

   We recommend to store flow cells unopened, as follows:

   Flow cell	Storage instructions
   Flongle Flow Cells	2–8°C for four weeks
   MinION/GridION Flow Cells	room temperature for one month, or at 2–8°C for 12 weeks
   PromethION Flow Cells	2–8°C for 12 weeks

1. Important In this step, you will use the MinKNOW software to check the number of pores in your flow cell. This has to be done as soon as reasonably possible after receiving your Flongle flow cell. Flow cells can then be stored until required. If there is a delay between receiving and using the flow cells, another check should be performed before library preparation begins.
2. How it works

   Flow cells are shipped with a QC DNA molecule present in the buffer. This molecule produces a distinctive nanopore signal. The MinKNOW™ software uses this signal to validate the integrity of the nanopore array before use and provides the user with an estimate of the number of simultaneously available channels for the experiment.

3. Place the Flongle adapter into the MinION or one of the five GridION positions.

   The adapter should sit evenly and flat on the MinION Mk1B or GridION platform. This ensures the flow cell assembly is flat during the next stage.

4. Important The adapter needs to be plugged into your device, and the device should be plugged in and powered on before inserting the Flongle flow cell.

   

5. Place the flow cell into the Flongle adapter, and press the flow cell down until you hear a click.

   The flow cell should sit evenly and flat inside the adapter, to avoid any bubbles forming inside the fluidic compartments.

   

6. Double-click the MinKNOW icon located on the desktop to open the MinKNOW UI.

   

7. 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.

   

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

   

   

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

8. Click the sequencing device connected to the computer.

   

9. The Sequencing Overview should show the flow cell has not had any checks carried out.

   Below is an example of a GridION device with a Flongle inserted into position X1 and MinION Flow Cells in positions X2 - X5.

   

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

    

11. Choose the flow cell type from the drop down menu and fill in the flow cell ID.

    Below is an example of a GridION device with a Flongle inserted into position X1.

    

12. 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 blox below:

    

13. Select 'Start' to begin the flow cell check.
14. You will be automatically navigated to the Sequencing Overview page.

    A loading bar appears under the flow cell during the checks.

    This example is a Flongle CTC (loading bar highlighted in red).
    

15. The flow cell check should take a few minutes.
16. Flow cell health indicators

    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.

    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 (question mark, exclamation mark or tick) will only remain visible during a MinKNOW session. Once the MinKNOW service has ended, the status of the flow cell will be erased.

1. In this step, you will use the MinKNOW software to check the number of pores in your flow cell. This has to be done prior to loading your DNA or RNA library onto the flow cell.
2. How it works

   Flow cells are shipped with a QC DNA molecule present in the buffer. This molecule produces a distinctive nanopore signal. The MinKNOW™ software uses this signal to validate the integrity of the nanopore array before use and provides the user with an estimate of the number of simultaneously available channels for the experiment.

3. Set up the MinION Mk1C and flow cell.

   Power on the MinION Mk1C, as described in the MinION Mk1C protocol under Power.

4. Once successfully plugged in, you will see a light and hear the fan.
5. Open the MinION Mk1C lid and insert the flow cell under the clip.

   Press down firmly to ensure good thermal and electrical contact.

   

   Note: The above image uses a CTC for illustrative purposes. Please insert a flow cell.

6. 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.

   

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

   

   

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

7. Select the Mk1C in the connection manager to open the device.

   

8. Navigate to the Start page and select 'Flow Cell Check' to open the flow cell check page.

   

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

   

10. The user will be automatically navigated to the Sequencing Overview page.

    Run state can be viewed here and a progress bar is displayed at the base of the flow cell.
    

    Number of pores available will be displayed here once check is completed.
    

11. The flow cell check should take a few minutes.
12. 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.

    

    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.

1. In this step, you will use the MinKNOW software to check the number of pores in your flow cell. This has to be done prior to loading your DNA or RNA library onto the flow cell.
2. How it works

   Flow cells are shipped with a QC DNA molecule present in the buffer. This molecule produces a distinctive nanopore signal. The MinKNOW™ software uses this signal to validate the integrity of the nanopore array before use and provides the user with an estimate of the number of simultaneously available channels for the experiment.

3. Open the MinION Mk1B/GridION lid and insert the MinION Flow Cell.

   Insert the flow cell in the MinION Mk1B/GridION by sliding the flow cell under the clip. Firmly press down on the flow cell to ensure good thermal and electrical contact.

   

4. Connect the assembled MinION Mk1B and flow cell to the host computer.

   

   Figure SSFC: Assembled MinION Mk1B and MinION SpotON Flow Cell connected to through the USB 3.0 port to the host computer

5. Once successfully plugged in, you will see a light and hear the fan.
6. Double-click the MinKNOW icon located on the desktop to open the MinKNOW UI.

   

7. 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.

   

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

   

   

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

8. Click the sequencing device connected to the computer.

   

9. The Sequencing Overview should show the flow cell has not had any checks carried out.

   Below is an example of a GridION device with a Flongle inserted into position X1 and MinION Flow Cells in positions X2 - X5.

   

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

    

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

    

12. You will be automatically navigated to the Sequencing Overview page.

    A loading bar appears under the flow cell during the checks.

    This example is a Flongle CTC (loading bar highlighted in red).
    

13. The flow cell check should take a few minutes.
14. Flow cell health indicators

    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.

1. How it works

   Flow cells are shipped with a QC DNA molecule present in the buffer. This molecule produces a distinctive nanopore signal. The PromethION software uses this signal to validate the integrity of the nanopore array before use and provides the user with an estimate of the number of simultaneously available channels for the experiment. Active pores are reported in four groups, each of which may be used in turn when running long experiments e.g. a 64 hour sequencing run. Shorter experiments may not use all groups.

2. Important After taking flow cells out of the fridge, wait 20 minutes before inserting the flow cell into the PromethION for the flow cell to come to room temperature. Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
3. Load the flow cells to be run into the docking ports of the PromethION.

   Insert the chip connector fully into the docking port. Take note to ensure the latch to hold the chip in place clicks in.

4. Important Correct insertion of PromethION flow cells into a PromethION 24/48 device

   Line up the flow cell with the connector (horizontally and vertically) and smoothly insert it into position.

   

   Press the flow cell down firmly into the loader, and ensure that the latch engages.

   

   Insertion of the flow cell at the wrong angle can damage the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position damaged, please contact support@nanoporetech.com for assistance.

   

   Once the flow cells are correctly loaded into the PromethION, the flow cells undergo an automatic hardware check:

   • Green tick: flow cell has passed
   • Red tick: flow cell needs to be re-inserted
5. Double-click the MinKNOW icon located on the desktop to open the MinKNOW UI.

   

6. 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.

   

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

   

   

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

7. Click the sequencing device connected to the computer.

   

8. The Sequencing Overview page should show the flow cell has not had any checks carried out.

   

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

   

10. When you see the flow cell type and flow cell IDs recognised, click Start to begin.

    

11. You will be automatically navigated to the Sequencing Overview page.

    A loading bar appears under the flow cell during the checks.

    This example is a Flongle CTC (loading bar highlighted in red).
    

12. The flow cell check should take a few minutes.
13. Flow cell health indicators

    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.