Nucleic Acids Research, Vol 26, Issue 14 3404-3409, Copyright © 1998 by Oxford University Press
JK Bonfield, C Rada and R Staden
The final step in the detection of mutations is to determine the sequence
of the suspected mutant and to compare it with that of the wild-type, and
for this fluorescence-based sequencing instruments are widely used. We
describe some simple algorithms forcomparing sequence traces which, as part
of our sequence assembly and analysis package, are proving useful for the
discovery of mutations and which may also help to identify misplaced
readings in sequence assembly projects. The mutations can be detected
automatically by a new program called TRACE_DIFF and new types of trace
display in our program GAP4 greatly simplify visual checking of the
assigned changes. To assess the accuracy of the automatic mutation
detection algorithm we analysed 214 sequence readings from hypermutating
DNA comprising a total of 108 497 bases. After the readings were assembled
there were 1232 base differences, including 392 Ns and 166 alignment
characters. Visual inspection of the traces established that of the 1232
differences, 353 were real mutations while the rest were due to base
calling errors. The TRACE_DIFF algorithm automatically identified all but
36, with 28 false positives. Further information about the software can be
obtained from http://www.mrc-lmb.cam.ac.uk/pubseq/
ARTICLES
Automated detection of point mutations using fluorescent sequence trace subtraction
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
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