DNA extraction and sequencing
tumor
DISEASE PROGRESSION, TUMOR, BLOOD
To explore potential biomarkers for response and resistance, patient blood samples were collected before treatment, on treatment (C2D1) and after disease progression. Blood samples were centrifuged to separate peripheral blood lymphocytes (PBLs) from plasma. Genomic DNA (gDNA) from PBLs was extracted using the RelaxGene Blood DNA System (TianGen Biotech Co., Ltd., Beijing, China). Cell-free DNA (cfDNA) and circulating tumor DNA(ctDNA) were extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany). Isolated gDNA was sheared by dsDNA fragmentase (New England Biolabs, Ipswich, MA, USA). DNA fragments were selected based on size using AMPure XP beads (Beckman Coulter, Inc., Brea, CA, USA) and libraries were constructed using the KAPA Library Preparation Kit (Kapa Biosystems, Inc., Wilmington, MA, USA). Agencourt AMPure XP beads (Beckman Coulter, Inc.) were used for all clean-up steps. End repair and 3′-end A-tailing, PCR amplification, ligation and single-step size selection were performed following DNA fragmentation.Targeted capture was performed using a custom set of biotinylated DNA probes, which cover 107 cancer-related genes (HapOncoCDx™, Roche). Amplified sample libraries and the SeqCap EZ Library were hybridized according to the manufacturer’s protocol. Subsequently, the reactions were pooled and purified using Agencourt AMPure XP beads, and then amplified by PCR. After quantification by quantitative PCR, the library was diluted, denatured with 0.2 N NaOH, and sequenced using PE150 paired-end sequencing on the NovaSeq 6000 system (Illumina, Inc., San Diego, CA, USA).DNA fragments, library purity and concentration were assessed using a Qubit 3.0 Fluorometer and dsDNA HS Assay kit (Invitrogen, Waltham, MA, United States). Fragment length was determined on a 4200 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cfDNA-seq was performed using a targeted next-generation sequencing panel on 107 lung cancer-related genes (HapOnco™ 107 panel) with a mean coverage of 2000×, and the mean coverage for gDNA is 1000×. Genes and alterations included in the HapOnco™ 107 panel are listed in the Supplementary Tab 