Mutation Detection

Filename

trace_diff.p4m

Description

This module compares each sequence chromatogram against a "wild type" or reference chromatogram to detect point mutations. The wild type file may be any normal trace from a standard comparator sequence or may be produced by averaging the traces of all the chromatograms (which can presently only be done in Gap4, thus requiring two runs of Pregap4 and Gap4). The mutations are detected by aligning and subtracting each trace from the wild type trace to produce a "difference trace". The difference trace is then analysed to identify point mutations which are written back to the Experiment File and MUTN tags. This uses the trace_diff program (see section Introduction to mutation detection). Bonfield, J.K., Rada, C. and Staden, R. Automated detection of point mutations using fluorescent sequence trace subtraction. Nucleic Acids Res. 26, 3404-3409 (1998).

Option: Wild type file

This is the filename of the chromatogram for the wild type sequence. This may be in SCF, ABI or ALF format.

Option: Start position

Option: End position

These define the range within each sequence in which to identify mutations. The algorithm works better on good quality data so including very bad sequence may give errors.

Option: Score

This a threshold used to determine when a peak in the difference trace is considered to be a mutation. The higher the value the more stringent the test.

Option: Alignment band width

The trace alignment is performed by firstly doing a sequence alignment on the text sequences contained in the two files. This specifies the band width for this alignment. Smaller values give quicker alignments, but only work if the alignment is sufficiently close to the main diagonal.

Option: Other arguments

This allows for any other arguments to be passed to the trace_diff program. See the trace_diff documentation for more details.