Qiagen PyroMark Q24 MDx, User Manual

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Appendix B
PyroMark Q24 MDx User Manual 01/2016 B-15
Quality assessment
The user will be warned if something in the assay may
reduce the quality of the result given by the analysis software.
The ultimate goal for a well-optimized assay is that all
variable positions in an AQ or CpG assay, or the sequence
in the quality control window of a SQA assay, have the
quality assessment “Passed” when using default or more
stringent analysis parameters. Such results will be shown as
blue in the quality bar in the well after analysis. Results of
lower quality are indicated as “Check” (yellow) or “Failed”
(red) together with error messages.
Analysis results
For samples and positive controls, aim for:

Sufficient signal intensities. Aim for a single peak height
of at least 40 RLU

No background in blank dispensations

No background in variable positions (AQ and CpG)

Expected reference sequence pattern (AQ and CpG)

All positions (AQ and CpG) and quality control window
(SQA) with quality assessment “Passed”.
The quality assessments for AQ and CpG assays are based
on the sequence context as well as the results in the analyzed
positions. Deviations from this built-in quality control are
shown as warnings in the “Well Information” area.
The analysis results for SQA assays are based on the
appearance of the peaks in Pyrogram, related to peak height
levels estimated by the software.
The inclusion of known bases in SQA assays can improve the
estimation of peak height level.