Backtracking of RAS pathway mutations by ultra-deep sequencing. A total of 22 mutations (out of 28) were detected in matched diagnosis samples. The 12 mutations (solid grey) determined by Sanger sequencing were detected at high mutant allele frequency ranging from 22% to 42%. Ten mutations (diagonally striped) were missed by Sanger sequencing but detected by ultra-deep sequencing. These mutations showed a low mutant allele frequency varying from 0.3% to 8%.