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Basecalling overview
A user can basecall and demultiplex their data directly in MinKNOW after a sequencing experiment has finished to avoid having to use command-line tools, or to re-analyse old data using the latest basecalling models. Reads can also be aligned against a reference post-sequencing.
Note: Both barcoding and alignment can be run on FASTQ, POD5 or FAST5 reads when coupled with basecalling.
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Click 'Analysis' on the start page.
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Click 'Basecalling' to open the post-run basecalling set-up options.
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Select input folder containing the FAST5 files to basecall from a previous run.
Select whether you want to process FAST5 reads in sub-directories.
If your chosen read input folder contains sub-directories with FAST5 files, you can choose whether to basecall FAST5 files in these sub-directories by enabling this option.
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Select output folder and file type.
By default, MinKNOW will create a
/basecalled
folder in/data
. You can set a different folder in which to save the basecalled reads.
Note: This must be a sub-folder of/data
directory.Select whether to output FAST5 files. If selected, these files will be written to a folder within
/basecalled
called/workspace
, if selected.Users are also able to enable read splitting options post-run.
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Optional actionSelect to use alignment and import a reference sequence in a FASTA file.
We currently only recommend uploading an alignment reference locally for bacterial-sized genomes. Upload can take a few minutes and is compute dependent.
A reference file must by uploaded as a FASTA file that can contain multiple entries in the same file (e.g. multiple chromosomes).
However, a .bed file may also be uploaded alongside the reference FASTA file. The BED file option can be used when the user is interested in a particular region of the reference (e.g. specific gene in the chromosome).
Alignment generates FASTQ output files when performed with basecalling. -
Click 'Start' to begin post-run basecalling.