L220 sonicator

NGS panel sequencing and analysis were performed at the Genomics Laboratory of GenomicCare Biotechnology (Shanghai, China). For the FFPE tissue, DNA was extracted using the MagMAX FFPE DNA/RNA Ultra Kit (cat# A31881, Thermo Fisher, Waltham, MA, USA) on a KingFisher Flex System (Thermo Fisher). The extracted DNA was sheared using a Covaris L220 sonicator, and then library preparation and capture were performed using a Tecan EVO 150 (Thermo Fisher), and the DNA was sequenced on an Ion S5 sequencer (Thermo Fisher) to generate paired-end reads. After removing adapters and low-quality reads, the reads were aligned to the National Center for Biotechnology Information (NCBI) human genome reference assembly hg19 using the Burrows-Wheeler Aligner algorithm and further processed using the Genome Analysis Toolkit (GATK, version 3.5), including the GATK Realigner Target Creator to identify regions that needed to be realigned. Somatic single-nucleotide variants (SNVs), insertions and deletions (indels), and copy number variations (CNVs) were determined using MuTect/ANNOVAR/dbNSFP31, VarscanIndel, and CNVnator software, respectively, as reported in Zang et al. [12] .

NGS panel sequencing and analysis were performed at the Genomics Laboratory of GenomicCare Biotechnology (Shanghai, China). For the FFPE tissue, DNA was extracted using the MagMAX FFPE DNA/RNA Ultra Kit (cat# A31881, Thermo Fisher, Waltham, MA, USA) on a KingFisher Flex System (Thermo Fisher). The extracted DNA was sheared using a Covaris L220 sonicator, and then library preparation and capture were performed using a Tecan EVO 150 (Thermo Fisher), and the DNA was sequenced on an Ion S5 sequencer (Thermo Fisher) to generate paired-end reads. After removing adapters and low-quality reads, the reads were aligned to the National Center for Biotechnology Information (NCBI) human genome reference assembly hg19 using the Burrows-Wheeler Aligner algorithm and further processed using the Genome Analysis Toolkit (GATK, version 3.5), including the GATK Realigner Target Creator to identify regions that needed to be realigned. Somatic single-nucleotide variants (SNVs), insertions and deletions (indels), and copy number variations (CNVs) were determined using MuTect/ANNOVAR/dbNSFP31, VarscanIndel, and CNVnator software, respectively, as reported in Zang et al. [12] .