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Calibration Strand detection
The DNA calibration strand (DCS) is a 3.6 kb amplicon of the Lambda phage genome. The RNA calibration strand (RCS) is the 1.4 kb enolase 2 (ENO2) gene transcript.
Calibration strands are added to DNA/RNA samples during library preparation, and are processed and included in the library.Detection of calibration strands by the basecaller can be used to assess how well basecalling has worked, and to confirm that the sample preparation was successful.
If
--calib_detect
is enabled, Guppy will attempt to identify and analyse any calibration strands which have been basecalled. Specifically, Guppy detects the calibration strand that is associated with the basecall configuration being used
(e.g. in experiments running an RNA configuration it looks for the RNA CS, and for DNA configurations it looks for DNA CS). It does this by first checking to see if a basecalled strand is approximately the correct length (controlled by--calib_min_sequence_length
and--calib_max_sequence_length
), and it then aligns the basecalled strand to the calibration strand reference. Successfully aligned reads are placed in thecalibration_strands
folder, and alignment accuracy and identity metrics are added to thesequencing_summary.txt
file. This can be a useful way of evaluating the quality of a run.