Trace_diff calculates the mean and the standard deviation of the difference trace. It then scans the difference trace to find bases for which the difference trace amplitude is more than n standard deviation units above the mean. These bases are tagged as mutants. The value of n must be supplied by the user and can only be determined by trial and error as it depends on the machine, chemistry, user, etc. For our work with dye terminators we found that n = 4 was suitable although some weak G after A mutations were missed, but for BigDye terminators n = 3 was best. Obviously, for any given reference trace and set of conditions these numbers only need to be chosen once and can then be used for all subsequent experiments. Note that n is a real number, not an integer, and so can be given values such as 3.75. If the results from trace_diff are checked using the gap4 contig editor, the performance using any given value of n can be monitored by counting the numbers of false positives and false negatives, and adjusting accordingly. Another method of quality control would be to always include the DNA from a suitable known mutant with each batch of samples. The sd values obtained for the known samples could then be checked to see if they were in the expected range.

URL: http://www.mrc-lmb.cam.ac.uk/pubseq/manual/mutations_4.html