This module compares each input sequence chromatagram against a wild type or
reference chromatogram and itself to detect heterozygote mutations. Heterozygote
mutations are detected by examining the chromatogram for double overlapping
peaks. A mutation "mask" is established by a set of double peak parameters. Comparisons
are made against this mask to determine if the double peak in question is a valid
Most of these parameters are user definable since no one set of parameters will be
suitable for all data. The default parameters have been chosen to maximise heterozygote
detection and at the same time, minimise the number of false positives reported. Some
experimentation may be required to optimise these for your own data. After
chromatagram analysis has been completed, mutation tags are written back to the
Experiment File as
Both the wild type and the input traces are scanned for mutations. Any mutation
appearing on the wild type and the input trace is ignored by the Heterozygote Scanner.
This helps to cut down on the number of false positives reported. The reference or
wild type traces for hetscan are specified in the
see section Reference Traces and Reference Sequences.
- Option: Maximum peak alignment deviation
The centres of each individual half-peak of a double peak must align reasonably
well for them to be considered to be real heterozygotes. The amount of half-peak
alignment deviation allowable is specified in bases by this parameter, usually
as a fraction of one base.
- Option: Minimum amplitude for peak 1|2
Low level noise in a trace can often look like perfect double peak heterozygotes
to the heterozygote scanner. These options setup a mask for the peak amplitudes
as a percentage of the maximum trace value. Anything below these percentages isn't
considered to be a valid heterozygote. Peak 1 refers to the highest amplitude
peak, whereas peak 2 refers to the 2nd highest amplitude peak.
- Option: Minimum peak amplitude ratio
The ratio of peak 1 to peak 2 amplitude is used to help identify true heterozyotes
from noise and gel artifacts. This parameter forms a mask such that any double peak
whose amplitude ratio is less than this value is rejected by the heterozygote scanner.
Normally you'd expect the ratio to be in the region of 0.5, but trace preprocessing
can distort these figures somewhat.
- Option: Complement bases on reverse strand tags
After mutation detection and after readings have been assembled into a GAP4
database, GAP4 displays both forward and reverse readings in a single direction
in the contig editor. This makes it much easier to compare sequences and traces
in both directions simultaneously. When the corresponding traces are displayed,
any reverse strand traces are complemented automatically such that the bases are
interchanged. In this case, the original mutation tag generated by hetscan will
then be of the wrong sense, so if checked, this option complements the tag base
labels to match the complemented trace displayed by GAP4.
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Last generated on 22 October 2002.