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Introduction to live basecalling in MinKNOW
For MinION Mk1B and Flongle (on MinION Mk1B), the MinKNOW software presents an option to basecall reads on the local computer. The basecalling is carried out live, as the read files are generated during a sequencing experiment.
Basecalling results are displayed in real-time in the MinKNOW user interface, and data is written out in the BAM or FASTQ file format.
The MinION Mk1C, GridION and PromethION devices have the capacity to perform live basecalling, while keeping up with one (MinION Mk1C), five (GridION) and up to 41 flow cells (on the PromethION 48, or 48 flow cells on PromethION 48 A100) running fast basecalling simultaneously. Basecalling is carried out directly on the device and uses the MinKNOW software. The basecalled reads are written to the device; users can then export the reads out for further analysis via the Network or removable storage.
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Live alignment in MinKNOW
Basecalled reads can be aligned to a reference during the sequencing run. To do this, you will need to upload a reference FASTA or .mmi file during run set-up, and optionally a BED file when there is a specific interest in a particular region of the reference (e.g. specific gene in a chromosome).
A reference file can contain multiple entries in the same file (e.g. multiple chromosomes), and alignment hits from these files are used to populate the alignment graphs which can be viewed on the MinKNOW UI. Alignment hits from BED files will appear in the sequencing .txt file generated in the data folder.