Qiagen PyroMark Q96 ID User Manual page 102

Appendix B
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, 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 Ts (read as Gs and As 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
Based on experience sequencing large numbers of
templates, the dispensation order n(CTGA) gives, on
average, the best de novo sequencing quality. Individual
templates may, however, give better results with other
dispensation orders.
SQA assays set up to differentiate multiple sequences should
not be designed to distinguish variants based 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
one target sequence in the sample. The assay may otherwise
generate a mixed sequence that cannot be analyzed.
B-6 PyroMark Q96 ID User Manual 01/2016