Mutation Detection

Description

Superceded by the newer modules: ( see section Trace Difference) and ( see section Heterozygote Scanner). This module compares each sequence chromatogram against a "wild type" or reference chromatogram to detect point mutations. 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 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). Obviously the reference traces should be as similar as possible to the ones being compared against it. It should be prepared by sequencing the wild type from the same primer, and using the same chemistry as the readings being screened. One good way to produce a reference trace is to run the wild type sequence on the gel along with the other samples. It is also possible to get gap4 to produce a consensus trace. This requires using pregap4 twice. Firstly process the sequences through pregap4 with all the appropriate options except with the mutation detection module disabled. Assemble these sequences into gap4. Within gap4, for each contig start up the Contig Editor and select Save Consensus Trace from the command menu. This will produce a trace which is the average of the traces in that contig. Then delete the gap4 database and reprocess the sequences using Pregap4, this time using mutation detection to compare against the consensus trace.

Option: Wild type file (+ve strand)

Option: Wild type file (-ve strand)

These are the filenames of the chromatogram for the wild type sequence on each strand. These may be in any allow trace format (SCF, ZTR, ABI, CTF or ALF). In the augment stage, these are represented in the WT line type using plus_filename|minus_filename notation.

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 parameter 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.

The module above is superceded by the newer modules: (see section Trace Difference) and (see section Heterozygote Scanner).