Whole-exome sequencing and exome analysis pipeline.

Genomic DNA was extracted from fresh-frozen tumor tissues and normal tissues (tails) with the use of a QIAamp DNA Mini Kit (QIAGEN) with RNase A (QIAGEN). DNA quality control was performed by agarose gel electrophoresis, and the purity of the DNA was analyzed by NanoDrop 2000 (Thermo Fisher Scientific). The purified genomic DNA was quantified by Picogreen (Thermo Fisher Scientific), and a minimum of 1 μg of DNA was used for whole-exome library construction. Whole-exome capture libraries were constructed with the use of Sure-Select Mouse All Exon, version 6.0 (Agilent Technologies). The exome libraries were quantified by real-time PCR using a Kapa Library Quantification Kit (Kapa Biosystems), and library quality control was run on the Agilent Bioanalyzer 2100 (Agilent Technologies). The constructed libraries were sequenced using an Illumina NovaSeq 6000 sequencer, yielding an average of 59 million reads (8.8 gigabases). The mean depths of target regions were 65.6–101.5. Sequencing reads were aligned to the GRCm38 (mm10) mouse genome assembly using the Burrows-Wheeler Alignment (BWA) tool, version 0.7.12. Duplicate reads were then identified and removed by using the Picard MarkDuplicates tool,version 1.130, and single-nucleotide and indel variants were called using MuTect2 of Genome Analysis Toolkit (GATK), version 3.8. The called variants were annotated by SnpEff tool, version 4.1. Variants were filtered out if their total reads in tumor or normal samples were less than 10, their variant reads in tumor samples were less than 3, their variant allele frequencies in tumor samples were less than 10%, and their allele frequencies in normal tissues were greater than 3%. Accepted variants that were nonsynonymous were counted as nonsynonymous TMBs.