Whole-genome sequencing was performed as described previously41. In brief, short-insert 500-bp genomic libraries were constructed according to Illumina library protocols and 100-base paired-end sequencing was performed on HiSeq 2000 or HiSeq X genome analysers to an average of 20× coverage. Short-insert paired-end reads were aligned to the reference mouse genome (NCBIM38) using BWA-MEM (http://bio-bwa.sourceforge.net/). For each HSC clone, the matched tail sample was used as the reference. We sequenced two of the Aldh2−/−Fancd2−/− HSC clones to 40× coverage, together with their matched germ-line references. We noted a big overlap between the variants found at 20× and 40× coverage (data not shown), showing that doubling the coverage did not actually uncover many more mutations. Any additional calls found when the coverage was increased to 40× were predominantly subclonal, with a mean VAF of 0.22. Therefore, we concluded that 20× whole-genome sequencing provided sufficient coverage to allow us to uncover mutations present in the transplanted HSC.
