Exome Sequencing of Gl261 Tumor Tissue

DNA from Gl261 tumor tissue from R and NR mice was extracted using the INVISORB^® DNA Tissue Mini Kit (STRATEC Biomedical AG) according to the manufacturer’s instruction. RNA contamination was eliminated by RNase digestion with 10 mg ml⁻¹ RNase at room temperature (RT) for 5 min (Sigma-Aldrich). Exome sequencing was performed on the Illumina NextSeq500 platform (Illumina Inc, San Diego, Calif.) using High output flow cell (75 nt reads paired end + 8 nt index). SureSelectXT Target Enrichment System (Agilent Technologies) was used for library generation according to the manufacturer’s instructions. To convert the vendor-specific sequencing data format generated by the Illumina NextSeq500 to a standard file format, the Illumina tool bcl2fastq (v2.15.0.4)⁴⁶ was used. To check the sequencing read quality, reports were generated with the tool fastqc (v0.10.1)⁴⁷ . After quality checks the alignment was performed with bwa mem (v0.7.5)⁴⁸ and the mouse reference genome GRCm38.68. The picard-tools (v1.105)⁴⁹ were used to remove duplicates from the alignment files. The sorting and indexing of these files was done with samtools (v0.1.19)^{50 (link)}. Afterward the variants were called by samtools mpileup (v0.1.19) for single-nucleotide variants and platypus (v0.7.9.1)⁵¹ for insertions and deletions. The basic annotations of the called variants was done with annovar (v2013-08-23)^{52 (link)}.