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How to start sequencing
Once you have loaded your flow cell, the sequencing run can be started on MinKNOW, our sequencing software that controls the device, data acquisition and real-time basecalling.
It is assumed you have already installed MinKNOW on your computer or device. Further instructions for setting up a sequencing run can be found in the MinKNOW protocol.
Please ensure when setting up a sequencing run you are using the recommendations outlined below. All other parameters can be left to their default settings.
MinKNOW can be used and set up to sequence in multiple ways:
- On a computer either direcly or remotely connected to a sequencing device.
- Directly on a PromethION 24/48 sequencing device.
For more information on using MinKNOW on a sequencing device, please see the device user manuals:
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Open the MinKNOW software using the desktop shortcut and log into the MinKNOW software using your Community credentials.
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Click on your connected device.
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Set up a sequencing run by clicking Start sequencing.
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Select the Ligation Sequencing Kit V14 (SQK-LSK114).
An expansion kit does not need to be selected.
Click Continue to Run Options to continue.
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Set the run options to a 72 hour run length and 20 bp minimum read length.
Click Continue to basecalling to continue.
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Set up basecalling using the following parameters:
- Ensure the basecalling is switched ON.
- Next to "Models", click Edit options and choose High accuracy basecaller (HAC) from the drop-down menu.
- Ensure barcoding is OFF.
Click Continue to output and continue.
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Keep the output format and filtering options to their default settings.
Click Continue to final review to continue.
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Click Start to start sequencing.
You will be automatically navigated to the Sequencing Overview page to monitor the sequencing run.
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Data analysis after sequencing
After sequencing has completed on MinKNOW, the flow cell can be reused or returned, as outlined in the Flow cell reuse and returns section.
After sequencing and basecalling, the data can be analysed, as outlined in the Downstream analysis section.