Sequencing analysis

We performed whole-exome sequencing with deep coverage (500×) to identify CH with recurrent somatic mutations (VAF > 0.02) using BM cells from irradiated male mice (N = 12). Among these mice, eight and four mice exhibited higher (20.9–15.1) and lower (14.8–14.2) RDW values, respectively, and were selected for sequencing analysis to investigate whether CH develops in irradiated mice with higher RDWs. Control male mice (N = 7) were also subjected to sequencing analysis. DNA was extracted from BM cells from the femur using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and sent to Macrogen, Inc. (Seoul, Korea) for library preparation using SureSelect XT Reagents (Agilent Technologies, Santa Clara, CA, USA) and sequencing on an Illumina NextSeq platform (San Diego, CA, USA). Sequence reads (fastq) were mapped using the Burrows–Wheeler alignment-maximal exact match algorithm to a mouse reference genome (mm10), after which the reads were sorted and indexed using SAMtools^15,16. Duplicate reads of polymerase chain reaction amplicons were identified and removed using the MarkDuplicates (Picard) tool¹⁷. Single-nucleotide variants (SNVs) or small insertions or deletions were identified using MuTect2, a GATK4 package¹⁷, using a non-irradiated control mouse as a reference. To confirm the results, variant calling was performed using our previous results of whole-genome sequencing of young adult C57BL6/C3H F1 mice (Biosample IDs, SAMD00164355, SAMD00164356, SAMD00164357, SAMD00164358, SAMD00164359, and SAMD00164360, which were registered in the DDBJ Sequence Read Archive; https://www.ddbj.nig.ac.jp/dra/index-e.html) as another reference. Suspected germline variants were excluded based on a high VAF of > 0.35. That is, only variants with a VAF of 0.02–0.35 were further evaluated^1,4. Variants supported by ≤ 5 reads or detected in most mice examined were filtered out to reduce artifacts. The effects of genetic variants were annotated and predicted using SnpEff¹⁸.

The frequencies of the detected somatic mutations were validated using the remaining DNA from BM cells by performing targeted amplicon sequencing (Illumina MiSeq, average depth of 30,000) with primers that amplified regions containing the mutated sequences. The Nextera XT Index Kit (Illumina) was used to attach dual indices and sequencing adaptors. The tissue specificities of the somatic mutations were also assessed in the BM, spleen, thymus, tail, kidneys, liver, brain, thyroid gland, and testes by performing targeted sequencing of DNA from these tissues.