Qiagen PyroMark Q24 MDx, User Manual

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Appendix B
PyroMark Q24 MDx User Manual 01/2016 B-13
the dispensation order with one blank dispensation and have
at least the same number of blank dispensations as the
number of variable positions.
Pay attention to tips and warnings indicated by the red
icon and make suitable modifications when the icon
appears.
Bisulfite treatment controls in CpG assays
When creating a CpG assay, inclusion of bisulfite treatment
controls is recommended. Cytosines not followed by a
Guanine, indicated as orange Ts in PyroMark ADSW, should
be fully converted to Thymine during bisulfite treatment and
can therefore be used as controls for the reaction. When
creating a CpG assay in PyroMark Q24 MDx Software, the
software indicates possible dispensations as controls for the
bisulfite treatment reaction. The original sequence (before
bisulfite treatment) can be entered in the assay and used to
see if the suggested controls are Cs converted to
T s (read as
Gs and A s in a reverse assay) and suitable as controls, or
not. The preferable controls are those dispensations that are
located at the beginning of the sequence and/or represent
single base incorporations.
SQA assays
Experience with sequencing large numbers of templates
indicates that the dispensation order n(CTGA) gives, on
average, the best sequencing quality. Individual templates
may, however, give better results with other dispensation
orders.
Where possible, SQA assays that involve resolution of
different sequences should be designed such that resolution
does not depend on accurate sequencing of homopolymers.
In addition, it may be useful to have a few known bases at
the beginning of the sequence, preferably single peaks.
These can be used as reference peaks to aid the setting of
the peak levels in difficult assays.
Ensure that the initial DNA sample is pure or that the assay is
capable of specifically amplifying and/or sequencing only