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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 GridION, MinION Mk1C or 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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Click the Native Barcoding Kit 24 V14 (SQK-NBD114.24).
Click Continue to Run Options to continue.
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Keep the run options to their default settings of 72 hour run length and 200 bp minimum read length.
Minimum read length can be reduced down to 20 bp to increase output by sequencing more short reads, such as contaminanting reads including ITR tetramers. During development of this protocol, it was noted that despite the increase in short reads, a higher proportion of short reads were of lower Qscore (≤9).
Click Continue to basecalling to continue.
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Set up basecalling and barcoding using the following parameters:
Ensure basecalling is ON.
Next to Models, click Edit options and choose the High accuracy basecaller (HAC) from the drop-down menu.
Ensure barcoding is ON and use the default settings.
A reference sequence may be uploaded to perform live alignment but this may slow down system processing.
Click Continue to output and continue.
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Set up the output format and filtering as follows:
Raw reads is on and select .POD5 as the output format.
Ensure .FASTQ is selected for basecalled reads.
Filtering is on and use the 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.