3_Whole Exome Sequencing
Flash frozen tumor samples were stored at -80C and shipped on dry ice to Novogene for whole exome sequencing. Genomic DNA from each sample underwent library preparation using the Illumina workflow.  DNA was sheared into fragments of 180-280 bp, followed by end repair, A-tailing, and Illumina adapter ligation.  Exon capture was achieved through hybridization with biotin-labeled probes and streptavidin-coated magnetic beads.  Unhybridized fragments were washed away, probes were digested, and the captured libraries were PCR amplified. Library quality was assessed using Qubit for quantification, and Bioanalyzer for size distribution analysis.  Libraries were then pooled based on concentration and sequencing requirements, and sequenced on the Illumina platform using a paired-end 150-cycle (PE150) strategy. Raw reads underwent stringent quality filtering.  Reads containing adapter contamination (more than 10 nucleotides aligning to the adapter sequence with ≤10% mismatches), those with >10% uncertain bases (N), and those with >50% low-quality bases (Phred quality <5) were removed.  Post-filtration data were assessed for sequencing error rate distribution), GC content distribution, and sequencing quality distribution to further ensure data integrity.  Clean reads were aligned to the human reference genome (hg38) using Burrows-Wheeler Aligner (BWA, v0.7.17).  Alignment files (BAM format) were sorted using Sambamba (v0.7.1), and duplicate reads were marked using Picard. Sequencing depth and coverage were calculated from the processed BAM files. Germline SNPs and InDels were called using GATK (v4.0).  Variant annotation was performed using ANNOVAR (2015Dec14). This included determining the genomic location of variants, predicting their functional consequences (e.g., synonymous, missense, frameshift, stopgain, stoploss), and assessing their potential pathogenicity using various prediction tools (SIFT, PolyPhen, MutationAssessor, LRT, CADD) and conservation scores (GERP++). Allele frequencies were obtained from several population databases, including 1000 Genomes Project, ExAC, gnomAD, and ESP.  Information from dbSNP, COSMIC, OMIM, GWAS Catalog, and HGMD was also incorporated.