BMC Genomics 2006, 7:73 http://www.biomedcentral.com/1471-2164/7/73 SFeigquernec2e features of a typical read Sequence features of a typical read. Sequence was gen- erated from the negative strand with the M13 Reverse primer (M13R). Exact matches to the wild-type and lox71 and lox66 inverted repeat mutants were required for spacers to proceed to further analysis. The NotI site, pUC19 vector boundary and EcoRI were also detectable. The HindIII site could not be detected with the M13 Reverse primer. sequence features (median feature location from read start given in bp): left wild-type inverted repeat (14 bp), first spacer (27 bp), right wild-type inverted repeat (34 bp), NotI site (65 bp), lox71 inverted repeat mutant (91 bp), second 8 bp spacer (104 bp), lox 66 inverted repeat mutant (111 bp), start of the pUC19 vector (133 bp) and EcoRI site (143 bp) (Figure 2). Successful recombination reactions were defined as those sequences that contained exact matches to the wild-type inverted repeat sequences flanking an 8 bp spacer (ATTACTTCGTATA NNNNNNNN TATACGAAGTTAT) and the lox66, lox71 inverted repeat mutations flanking an 8 bp spacer (TACCGTTCGTATA NNNNNNNN TATACGAACGGTA) (Figure 2). Five spac- ers lacked the central TA nucleotides but were retained in the analysis because they successfully recombined. There were 3,124 reverse strand sequence reads from suc- cessful recombination reactions that were used for further analysis. However, these sequences could not be analyzed as is. First, each spacer was reverse complemented to facil- itate comparisons with published loxP spacers as most spacers are published in the positive strand orientation [6,7]. Since each sequence read represented the final prod- uct of recombination, the spacer sequence of the original input LE and RE oligonucleotides had to be inferred based on the published location of the scissile bonds [22] and mechanism of recombination (Figure 3) [14-16]. For each recombination reaction, the input LE oligonucleotide spacer was defined as loxP spacer position 1 from the LE/ RE double inverted repeat mutant and positions 2–8 from the spacer with wild-type inverted repeats (Figure 3). Accordingly, the input RE oligonucleotide spacer was defined as position 1 from the wild-type inverted repeat loxP spacer sequence and positions 2–8 of the LE/RE dou- ble inverted repeat mutant (Figure 3). However, there remained a further layer of complexity in the data. Each recombinant DNA molecule derived from mismatching spacers gave rise to two pools of PCR prod- ucts from the same PCR reaction (Figure 1, steps 5 & 6), one pool derived from amplification of the positive strand of the initial recombinant molecule and another from the negative strand. From each of these two types of PCR products we sequenced the top, or positive strand. Since we could only observe the sequence of these final PCR products, we had to infer the sequence of input oligonu- cleotides using the established loxP recombination mech- anism [14-16]. We defined four categories of inferred input oligonucleotides based on the location of the mis- matched base(s) (Table 3) (Figure 4). The Type I class (Figure 4A) corresponded to identical spacers in the two input oligonucleotides that yielded a recombinant mole- cule with identical spacers. For this class, the PCR prod- ucts and sequence reads originating from the positive versus negative strand of the original recombinant mole- cule were identical. The Type II class (Figure 4B) corre- sponded to input oligonucleotides with discrepancies in positions 1 and/or 8 in their reacting spacers. For this class the input oligonucleotides can be unambiguously assigned as well because positions 1 and 8 are not exchanged between strands during recombination. The Type III class (Figure 4C) corresponded to input oligonu- cleotides with one or more mismatches in positions 2 through 7, which are the bases that undergo strand exchange during recombination. For Type III, the sequence of the input spacers could be inferred but the origin within the LE or RE oligonucleotides was ambigu- ous. Lastly, the Type IV class (Figure 4D) corresponded to input oligonucleotides that had mismatches in positions 1 or 8, and also had one or more mismatches in positions 2 through 7. For this class the precise identity of the input Page 5 of 13 (page number not for citation purposes)