Hiseq ngs platform

A NGS approach was performed on genomic DNA isolated from formalin‐fixed paraffin‐embedded (FFPE) surgically resected tumor samples (Fig 1c). Two commercial targeted pan‐cancer NGS panels were conducted by Gene⁺OncoMDR (1021 cancer related genes) and GeneseeqOne (416 cancer‐relevant genes) respectively, both of which were performed on the Hiseq NGS platforms (Illumina Inc., San Diego, CA, USA). TMB is defined as the rate of peptide‐changing single nucleotide variations (SNVs) per Mb. To estimate TMB of all tumors, SNVs with a mutation allelic fraction (MAF) of at least 0.1 after standard filtering and with high or moderate putative impact were retained. In a recent study, comprehensive analysis of more than 100 000 cases pan‐cancer genomes revealed that the median TMB in all cancers is 3.6.26 We therefore defined TMB > 4 as relatively high (TMB^high) and TMB ≤ 4 as low (TMB^low).

DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany. Purified DNA was qualified by Nanodrop One (Thermo Fisher, Waltham, MA) and quantified by Qubit 3.0 (Life Technologies, Singapore) using the dsDNA HS Assay Kit (Life Technologies, Eugene, OR) according to the manufacturer’s recommendations. Different libraries with unique indices were pooled together in desirable ratios for up to 2 μg of total library input. Human cot-1 DNA (Life Technologies, Carlsbad, CA) and xGen Universal blocking oligos (Integrated DNA Technologies, Coralville, IA) were added as blocking reagents. The capture reaction was performed with the NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche, Madison, WI) and Dynabeads M-270 (Life Technologies, Vilnius, Lithuania) with optimized manufacturers’ protocols. Captured libraries were on-beads amplified with Illumina p5 and p7 primers in KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Cape Town, South Africa). The post-capture amplified library was purified by Agencourt AMPure XP beads and quantified by qPCR using the KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by the Agilent Technologies 2100 Bioanalyzer (Agilent, Santa Clara, CA). The target-enriched library was then sequenced on HiSeq NGS platforms (Illumina, San Diego, CA) according to the manufacturer’s instructions.

Total RNA from FFPE samples was extracted using the RNeasy FFPE kit (QIAGEN). Total RNA amount was quantified by the Bioanalyzer 2100 (Agilent Technologies). Ribosomal RNA and residual genomic DNA were depleted by the KAPA Standard RNA-Seq Kit with RiboErase (HMR) and DNase digestion, followed by purification using the Agencourt RNA Clean XP Beads according to the manufacturers’ protocol. KAPA Stranded RNA-Seq Library Preparation Kit was used to construct Illumina-compatible sequencing libraries including RNA fragmentation and priming, double-stranded cDNA synthesis, adaptor ligation and PCR amplification.
Sequencing was performed on Illumina HiSeq NGS platforms (Illumina). To generate sequence reads in the FASTQ format, base calling was performed on bcl2fastq V.2.16.0.10 (Illumina) and quality control was performed with Trimmomatic (V.0.33).10 (link) RNA-seq reads were mapped to the human genome (hg19, Genome Reference Consortium GRCh37) using STAR (V.2.5.3a)13 (link) to identify individual exon, intron and intergenic features. The average coverage of mapped reads across base positions of the feature coordinates were calculated. Exon junctions were detected and visualised on the Integrative Genomics Viewer.14 (link)