We all love the MiSeq for the fast turnaround, remarkable data quality and its streamlined workflow. However, from time to time, our trusty machines will have an off day that can throw an elephant-sized wrench into your plans. From time to time, you may notice that you are unable to start your Miseq run because the pre-run flow check is failing.
Obviously, it’s imperative that you have good flow through your newly loaded flow cells for the Miseq to generate proper clusters and good data quality.
The Miseq needs to check and measure flow rate so that your equipment will know how much reagent it is pumping during the sequencing run. On the MiSeq, there are sensors that measure this flow. These sensors work by detecting the fluid in the line to make sure the correct volume is flowing through the machine. If your flow rate is off, your MiSeq will either over or under-pump reagents, negatively affecting your data quality.
Inspect the Flow Cell First
Thankfully, if your MiSeq pre-run flow check is not passing, there are a number of easy troubleshooting steps that can be taken that often resolve the issue with no maintenance or service to the system needed. First, open the MiSeq door and remove the flow cell. Clean the stage and flow cell using a wet lint-free wipe. It is a good idea to clean the stage even if no reagent buildup is visible. While the flow cell is removed from the system, inspect it for any signs of damage such as chips, cracks or scratches.
You'll also want to make sure the flow cell is clean, as a dirty flow cell will negatively affect data quality. You can clean it using a lint-free wipe. Return the flow cell to the stage, taking care that it is seated properly. Attempt another flow check. If the flow check still fails, you can manually force fluid through the properly seated flow cell by taking the following steps in the MiSeq Control Software:
- Select the following: Solution: PR2, Volume: 250, Aspiration Rate: 2500, Dispense Rate: 2500
- Select “Pump”
- Retry the flow check
If You Still Have Issues, Run a Power Cycle
If you try all of these steps and the flow check still fails, you will want to power cycle the instrument:
- To power cycle, close the MiSeq Control Software (MCS), shut down Windows, and switch off the instrument using the switch in the back of the unit
- Power back on the system using the switch on the rear and open MCS
- Attempt another flow check
- If the flow check still fails, run a maintenance wash and try the flow check again
If none of the previous troubleshooting steps were successful, record the lot number of the flow cell and try using a flow cell from another kit. For flow cells that fail to pass the flow check, you can contact the OEM to receive a replacement.
It can be frustrating, but these machines do break down from time to time. To eliminate random down days and keep your lab running at maximum efficiency, schedule a preventative maintenance visit on your MiSeq. One day with a SeqGen service technician can save you massive headaches down the road!