The two sequences are placed on the axes of a rectangular image and (subject to threshold conditions) wherever there is a similarity between the sequences a dot is placed on the image.
Where the two sequences have substantial regions of similarity, many dots align to form diagonal lines. It is therefore possible to see at a glance where there are local regions of similarity as these will have long diagonal lines. It is also easy to see other features such as repeats (which form parallel diagonal lines), and insertions or deletions (which form breaks or discontinuities in the diagonal lines).
dotmatcher uses a threshold to define whether a match is plotted (calculated from the substitution matrix). A window of specified length is moved up all possible diagonals and a score is calculated within each window for each position along the diagonals. The score is the sum of the comparisons of the two sequences using the given similarity matrix along the window. If the score is above the threshold, then a line is plotted on the image over the position of the window.
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For protein sequences EBLOSUM62 is used for the substitution matrix. For nucleotide sequence, EDNAFULL is used. Others can be specified.