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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:
To start a sequencing run on MinKNOW:
1. Navigate to the start page and click Start sequencing.
2. Fill in your experiment details, such as name and flow cell position and sample ID.
3. Select the Rapid Barcoding Kit 24 V14 (SQK-RBK114.24) or Rapid Barcoding Kit 96 V14 (SQK-RBK114.96).
4. Change the run limit to 12 hours by clicking "Options" and changing the run limit value to 12. The other run settings can be left at the defaults.
Note: We recommend a run-time of 12 hours for new users to ensure sufficient data is generated for downstream analysis. However, shorter run-times may be sufficient depending on your sample input and analysis requirements.
If using custom sequencing time, for optimal results, we recommend allowing the sequencing run to generate 1000-1500 reads per sample before proceeding to the analysis workflow.5. Set up basecalling and barcoding using the following parameters:
- Toggle basecalling to ON.
- Next to "Models", click Edit options and choose High accuracy basecaller (HAC) from the drop-down menu.
- Toggle barcoding to ON.
- Keep all other options at their default settings.
- Click Continue to output and continue.6. Set up the output format and filtering as follows:
- Select either .POD5 or .FAST5 (legacy) as the output format.
- Ensure .FASTQ is selected for basecalled reads.
- Ensure filtering is ON and read splitting is enabled. Other parameters can be kept to their default settings.
- Click Continue to final review to continue.7. Click Start on the Review page to start 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. For further information about options for basecalling and post-basecalling analysis, please refer to the Data Analysis document.
In the Downstream analysis section, we outline further options for analysing your data.