Bioinformatics pipeline and tumour mutation burden analysis

The bioinformatics pipeline and tumour mutation burden analyses were performed using the standard protocols mentioned previously [38]. The generated raw sequencing reads were filtered for adapter trimming and quality filtering using Trimmomatic. Obtained reads were aligned to the human genome reference hg19 through Burrows-Wheeler Aligner (BWA v0.7.12). SAM files were converted to sorted BAM files using NovoSort (v3.08.00). Genome Analysis Toolkit (GATK v3.7) was used to do local realignment around potential small insertions or deletions (indels) and base recalibration for next step mutation calling procedures, with duplicated reads removed. Then, we used VarDict (v1.5.1) to detect SNV and small indels, and FreeBayes (v1.1.0-44) was adopted to investigate complex mutations. Paired tumour normal sample calling was processed during the mutation calling. To filter out personal germline mutations, DNA translocation analysis was performed with FusionMap (v8.0.2.32). All base substitutions, short insertions, and deletions were initially recorded before filtering. The generated candidate mutations were annotated using Annovar software tools [39] and subsequently filtered using genomic databases such as Catalogue of Somatic Mutations in Cancer (COSMIC), the Short Genetic Variations database (dbSNP), and the Exome Aggregation Consortium (ExAC).

The TMB was defined as the number of somatic, coding, base substitutions, and indel mutations per megabases of the genome examined. All base substitutions and indels in the coding region of the targeted genes, including synonymous alterations, were initially counted before filtering as described above. Alterations that were predicted to be germline by the somatic—germline zygosity algorithm were not counted. Known germline alterations in dbSNP were not counted. Germline alterations occurring with two or more counts in the ExAC database were not counted [40]. To calculate the TMB per megabases, the total number of mutations counted was divided by the size of the coding region of the targeted territory. TMB was reported as a continuous variable. According to the TMB level, patients were divided into three groups: high, moderate, and low. The grouping criteria were based on the 75th percentile and 25th percentile of this batch of data. Then, a TMB level that was greater than or equal to the 75th percentile was defined as high. A TMB level less than the 25th percentile was defined as low, and the moderate level occurred between the 25th and 75th percentiles.