TMB counts were calculated as the number of synonymous and nonsynonymous mutations divided by the covered coding region. Polymorphisms/germline mutations identified by the TSO500 germline filter were evaluated by the comparison of multiple samples of the same tumor with varying tumor cell content and in nine cases by sequencing matched adjacent non-neoplastic lung tissue for nine specific cases in addition. NGS Data Analysis and TMB Determinationįurther, the presence of a variation called in one sample of a respective patient was checked in all associated samples. All assays were performed according to the manufacturers’ protocols. Up to eight libraries were sequenced simultaneously on a NextSeq 500 (Illumina) using high-output cartridge and v2 chemistry. Following two-target capture and purification steps, the enriched libraries were amplified (15 cycles polymerase chain reaction ) and subsequently quality controlled using the KAPA SYBR Library Quantification Kit on a StepOnePlus quantitative PCR system (both Thermo Fisher Scientific). To fragment the DNA strands to a length of 90 to 250 bp, 80 ng DNA of each sample were sheared according to their degradation level for 50 to 78 seconds using a focused ultrasonicator ME220 (Covaris, Woburn, Massachusetts). In the initial step of the library preparation for the capture-based TruSight Oncology 500 panel (Illumina), the grade of DNA integrity of a sample was assessed using the Genomic DNA ScreenTape Analysis on a 4150 TapeStation System (both Agilent, Santa Clara, California).
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