A limitation of our approach is that cells with the capacity to engraft may represent the least mutated HSCs. Nevertheless, we observed significant increases in the frequency of deletions, which were more prevalent (Fig. 5d, e) and larger (Fig. 5f) in Aldh2−/−Fancd2−/− genomes. The mean variant allele frequency (VAF) for all filtered indels was 0.47, establishing that these changes are of clonal origin. By examining the flanking regions, we found that microhomology-mediated deletions are the main contributors to the mutations observed in Aldh2−/−Fancd2−/− HSCs, indicative of NHEJ repair of double-stranded breaks23 (Fig. 5g, h). Additionally, the increase in the size of the deletions (Fig. 5f) suggests a role for alternative end-joining in the repair of some of these breaks, as alternative end-joining is characterized by increased resection in comparison to classical NHEJ24. Next, we analysed the location of indels across the genome, as recent work has suggested a role for the Fanconi anaemia pathway in preventing genomic instability at transcription–replication collisions25,26. However, we found no evidence of microhomology-mediated deletions being enriched at coding regions or transcribed genes (Fig. 5i, j), suggesting stochastic double-stranded break formation in Aldh2−/−Fancd2−/− HSCs.
