Figure 2

Cyclobutane Thymine Dimer (T=T) lesion and its detection by next generation sequencing. (a) Molecular scheme of the T=T-lesion with the cyclobutane ring formed with atoms C5 and C6 of the adjacent thymines. (b) Comparison of different hexamers containing an intact central TT sequence in comparison to the corresponding hexamers containing a T=T lesion at the same position. The test experiment clearly shows that the hexamers containing a T=T lesion show much smaller (more than 1000-fold) absolute frequencies than the intact hexamers. The large discrimination demonstrates that the proposed technique (Step 3 and 4) is well suited to detect the T=T damage by allowing only fully intact strands to be successfully sequenced. (c) Frequencies of the 65,536 sequences from a pool of DNA strands with eight central randomers (raw data). The synthesis of the sample, the preparation of the library and the sequencing result in sequence dependencies for the unexposed sample, which must be considered in the further analysis. Insert: Frequencies for a selection of sequences taken with increasing exposure (blue: unexposed, to green: maximum dose) showing sequence dependent decrease of the abundances.