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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. For more detailed information on setting up and using MinKNOW, please see the MinKNOW protocol.
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 200 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 baseceller (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.
However, .fast5 output can be used if you cannot use .pod5 output files.
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.