Sequencing frames and “bad” words in MPSS signatures. MPSS uses two sequencing reactions that are performed in reading frames shifted by two bases. These two frames are the 2-step or 4-step frames. Sequencing proceeds in sets of four bases (“words”). (A) The eight sequencing frames are indicated for the 17-base signatures, with the 2-step frames indicated below the example signature and the 4-step frames indicated above. The numbering 1 to 8 for the 17-base signatures is only used to illustrate the total number of words that are sequenced. The first sequencing reaction in the 2-step reaction always begins with TC. The three Ns in gray at the end of the 17-base signature indicate the bases that are not sequenced but that can affect the performance of the seventh or eighth word. (B) The 20-base signatures require an extra word (underlined “9”) that includes the two unsequenced bases indicated by the gray letter N. (C) The 20 “bad words” that underperform in MPSS sequencing. All 16 four-base palindromic words are listed at left; these words underperform in the MPSS sequencing reaction when they occur in the frames indicated for the 17- or 20-base signatures (see text). The four words in the box also underperform in the MPSS sequencing reaction, but these are not palindromic.
