Qiaamp dna formalin fixed paraffin embedded tissue kit

Tissue DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues using QIAamp DNA formalin-fixed paraffin-embedded tissue kit (Qiagen, Hilden, Germany). A minimum of 50 ng of DNA is required for NGS library construction. Tissue DNA was sheared using Covaris M220 (Covaris, MA, USA), followed by end repair, phosphorylation, and adapter ligation. Fragments between 200–400 bp from the sheared tissue DNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), followed by hybridization with capture probes baits, hybrid selection with magnetic beads, and PCR amplification. The quality and the size of the fragments were assessed using the Qubit 2.0 fluorometer with the dsDNA high-sensitivity assay kit (Life Technologies, Carlsbad, CA). Indexed samples were sequenced on Nextseq500 (Illumina, Inc., USA) with paired-end reads and average sequencing depth of 1000× using a panel with 520 cancer-related genes, spanning 1.64 megabases (Mb) of the human genome (OncoScreen Plus, Burning Rock Biotech, Guangzhou, China). The genes included in the panel are listed in Table S1.

DNA isolation and targeted sequencing were performed in Burning Rock Biotech, a commercial clinical laboratory accredited by the College of American Pathologist (CAP) and certified by the Clinical Laboratory Improvement Amendments (CLIA), according to optimized protocols as described previously [15 (link), 16 (link)]. Briefly, tissue DNA was extracted from formalin-fixed, paraffin-embedded tumor tissues using QIAamp DNA formalin-fixed paraffin-embedded tissue kit (Qiagen, Hilden, Germany) and circulating cell-free DNA (cfDNA) was extracted from 4–5 ml of plasma samples using a QIAamp Circulating Nucleic Acid kit, according to the manufacturer’s standard protocol (Qiagen, Hilden, Germany). Fragments between 200 and 400 bp from the sheared tissue DNA and cfDNA were purified (Agencourt AMPure XP Kit, Beckman Coulter, CA, USA), hybridized with capture probes baits, selected with magnetic beads, and amplified. Target capture was performed using a commercial panel consisting of 520 genes (OncoScreen Plus), spanning 1.64 megabases of the human genome. The quality and the size of the fragments were assessed by high sensitivity DNA kit using Bioanalyzer 2100 (Agilent Technologies, CA, USA). Indexed samples were sequenced on Nextseq 500 (Illumina, Inc., CA, USA) with paired-end reads and an average sequencing depth of 1,000 × for tissue samples and 10,000 × for liquid biopsy samples.

Frozen sections (10 μm in thickness) of tumor and nontumorous tissues were fixed in ice‐cold 4% formalin for 10 min, washed by water for 5 min, and subsequently stained by hematoxylin. Tumor and non‐tumorous cells were collected from the sections by laser microdissection (LMD) using LMD 7000 (Leica Microsystems, Inc., Bensheim, Germany). Genomic DNA was extracted from the collected cells using a QIAamp DNA formalin‐fixed, paraffin‐embedded tissue kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. Concentration of DNA was assessed by e‐SPECT (Malcom, Tokyo, Japan) and Qubit2 fluorometer (Invitrogen, CA). All genomic DNA was stored at −20°C until use.