Superceded by the newer modules:
see section Trace Difference)
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
Bonfield, J.K., Rada, C. and Staden, R. Automated detection of point
mutations using fluorescent sequence trace subtraction. Nucleic Acids Res. 26,
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
- 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
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).
This page is maintained by
Last generated on 22 October 2002.