Heterozygote Scanner

Description

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 heterozygote mutation. 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 HETE tags. 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 Traces --> 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.