FFPE samples containing at least 20% tumor cells [as determined from the examination of hematoxylin and eosin (H&E)-stained sections] were deparaffinized and genomic DNA (gDNA) was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany) in accordance with manufacturer’s instructions, followed by quantification using PicoGreen fluorescence assay (Invitrogen). The gDNA from white blood cell (WBC) samples was extracted using QIAamp DNA Blood Mini Kit (Qiagen) as described by the manufacturer.
All sequencing processes were accomplished in 3DMed Medical Laboratory Co., Ltd (Shanghai) (22 (link)). The details of NGS method are described in manuscript communicated for publication (Paper #NCOMMS-18-38299C). Illumina NextSeq 500 was used to sequence samples with the IDT xGen hybridization buffer. To evaluate the quality of the sequencing data, we used FastQC software (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/). BWA-MEM was used to map the sequence data to the human genome (hg19) reference. The results were sorted, and duplicate reads were removed with Picard (http://broadinstitute.github.io/picard/) (23 (link),24 (link)). In general, the mean sequencing depth of FFPE samples was 394× and that of matched blood samples was 431×.