Kapa htp library preparation kit for the platform

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens of the primary tumor from each patient were collected for analysis. The black PREP FFPE DNA Kit (Analytik Jena, Germany) was used to isolate DNA from the FFPE tissue specimens. Whole blood samples were centrifuged for 10 minutes (1,600 g) at room temperature to isolate lymphocytes, and a Tiangen Whole Blood DNA Kit (Tiangen, Beijing, China) was used to extract DNA from peripheral blood lymphocytes according to the manufacturer's instructions. Genomic DNA was sheared into 150-200 bp fragments with a Covaris M220 focused ultrasonicator (Covaris, Massachusetts, USA), and a DNA fragment library was constructed using a KAPA HTP Library Preparation Kit for the Illumina platform (KAPA Biosystems, Massachusetts, USA) according to the manufacturer's instructions. The DNA library was captured with a 543-gene plate designed based on the NimbleGen SeqCap EZ library (Roche, Wisconsin, USA), which includes key tumor-related genes. Captured samples were subjected to paired-end sequencing on the Illumina HiSeq X-Ten (cohort 1) or NovaSeq 6000 (cohort 2) platform.

Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from each patient’s primary tumor were collected for analysis. A blackPREP FFPE DNA Kit (Analytik Jena, Germany) was used to isolate DNA from the FFPE tissue specimens. Whole blood centrifuge (1,600 × g) for 10 min at room temperature was to isolate lymphocytes. A Tiangen Whole Blood DNA Kit (Tiangen, Beijing, China) was used to extract DNA from peripheral blood lymphocytes according to the manufacturer’s instructions. The genomic DNA was fragmented into 150–200-bp segments with a Covaris M220 focused ultrasonicator (Covaris, Massachusetts, USA). A fragmented DNA library was constructed with a KAPA HTP library preparation kit for the Illumina platform (KAPA Biosystems, Massachusetts, USA) according to the manufacturer’s instructions. DNA sequences from a DNA library (NimbleGen SeqCap EZ Library; Roche, Wisconsin, USA) including major tumor-associated genes, were captured with a 543-gene plate and subjected to paired-end sequencing with an Illumina HiSeq X-Ten instrument.

To identify somatic or germline single‐nucleotide variants (SNVs) and insertion and/or deletion (indel) mutations, FFPE tumor samples were used to detect mutations and matched blood samples were used as controls. A blackPREP FFPE DNA Kit (Analytik Jena, Germany) was used to perform DNA isolation from the FFPE segments. DNA of controls were extracted from peripheral blood lymphocytes by using Tiangen Whole Blood DNA Kit (Tiangen), and the lymphocytes come from Whole blood by centrifuging (1600g) for 10 min at room temperature. A Covaris M220 focused ultrasonicator (Covaris) was used for genomic DNA fragmentation (150–200‐bp segments) and a KAPA HTP library preparation kit for the Illumina platform (KAPA Biosystems) was used to construct the DNA library. Then the DNA library (NimbleGen SeqCap EZ Library; Roche) was captured with a 543‐gene panel involving the sequences of major tumor‐associated genes, and sequenced by an Illumina HiSeq X‐Ten instrument. These processes were all performed according to the manufacturers' instructions.