Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. However, such biases have not yet been measured in chromatin for lack of technologies. Here we develop a genome-wide assay whereby the same DNA lesion is repaired in different chromatin contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated when the single strand annealing repair pathway is used. Surprisingly, the mismatch repair machinery favors the same strand 60-80% of the time. The location of the lesion in the genome and the type of mismatch have little influence on the repair bias in this context. Using machine learning, we further show that both the repair bias and the efficiency of the repair are independent of known chromatin features. These results suggest that some intrinsic property of the lesion can have a large influence on the outcome of DNA repair, irrespective of the surrounding chromatin context.Recent insight into this question came from cancer genome sequencing (Pleasance et al . 2010;Hoadley et al . 2014) . In particular, this made it possible to show that the mismatch repair system in healthy cells is more accurate at some loci than others. For instance, mismatches in late-replicating regions are repaired less efficiently (Supek and Lehner 2015) , a feature that seems to be shared among eukaryotes (Weber, Pink, and Hurst 2012) . It is presently unknown whether the chromatin context can bias the mismatch repair toward one nucleotide or another, mostly because it is difficult to tease apart the contributions of DNA damage and DNA repair to mutation patterns.

Background Biases of DNA repair can shape the nucleotide landscape of genomes at evolutionary timescales. The molecular mechanisms of those biases are still poorly understood because it is difficult to isolate the contributions of DNA repair from those of DNA damage. Results Here, we develop a genome-wide assay whereby the same DNA lesion is repaired in different genomic contexts. We insert thousands of barcoded transposons carrying a reporter of DNA mismatch repair in the genome of mouse embryonic stem cells. Upon inducing a double-strand break between tandem repeats, a mismatch is generated if the break is repaired through single-strand annealing. The resolution of the mismatch showed a 60–80% bias in favor of the strand with the longest 3′ flap. The location of the lesion in the genome and the type of mismatch had little influence on the bias. Instead, we observe a complete reversal of the bias when the longest 3′ flap is moved to the opposite strand by changing the position of the double-strand break in the reporter. Conclusions These results suggest that the processing of the double-strand break has a major influence on the repair of mismatches during a single-strand annealing.