Figure 4

Schematics of Type I-IV mismatch reactions. Diagrammatic representation of how the four categories of input oligonucleotides were inferred. Recombination segments originating from the LE and RE oligonucleotides are highlighted with the same colour. Each initial recombinant molecule, when PCR amplified, produced two pools of double-stranded sequence; one from amplification of the positive strand and the other from amplification of the negative strand (Figure 1, steps 5 & 6). Since we sequenced and analyzed these PCR products in the 5'→3' orientation, rather than the actual recombinant molecule, we ensured that unambiguous input oligonucleotides were inferred for further analysis. A) Type I and B) Type II. These classes are defined as having either identical input spacers (Type I) or input spacers mismatched at positions 1 and/or 8 (Type II). In both types the original oligonucleotides could be inferred definitely. C) Type III. This class contains input spacers with different bases at positions 2,3,6 and 7. In all spacers positions 4,5 are fixed as TA during oligonucleotide synthesis. In these cases the spacer sequences could be identified but their origin within the LE or RE oligonucleotides was ambiguous. Regardless of initial oligonucleotide orientation, these mismatches resulted in two distinct recombinant molecules that gave rise to the same two pools of sequence reads. D) Type IV. This class contains input oligonucleotides with different bases at position 1 and/or 8 plus a mismatch at one or more of positions 2,3,6 and 7. In these cases the input spacer sequences are ambiguous as two possible recombinants arising from different sets of LE, RE oligonucleotides can give rise to the same pool of sequence reads. Mismatches between reacting spacers are indicated by red "X" marks in the input oligonucleotides