Figure 2.

Sequence motifs used to identify the orientation of each AK gene. MiSeq data contained four types of sequence reads for the P variants, including (A) two different types of sense-strand reads and (B) two different types of antisense-strand reads. Reads that occurred at the different ends of permuteposon either contained the start codon (green) or stop codon (red). These were designated Start and Stop motifs. (C) Unique 54 bp sequences were used to differentiate each type of sequence read in our analysis. After identifying whether a sequence read corresponded to the sense versus antisense strand (and Start or Stop motifs), the adjacent 11 bp sequence was compared with all possible 11 bp sequences within both the sense and antisense strands of the circular AK gene. This analysis allowed us to identify the orientation and sequence of the circularly permuted gene in the different sequence reads obtained from MiSeq analysis. The 5 bp sequence directly adjacent to the Start and Stop motifs was used to determine the AK residue at the beginning of each polypeptide.