Qubit 2.0 fluorometer with dsdna hs assay kits

Blood samples were collected in K3EDTA‐containing tubes (Cell‐Free DNA BCT) and centrifuged at 2000 g at 4°C for 10 min within 72 h of their collection. The carefully aspirated plasma supernatant was transferred into fresh 15 ml centrifuge tubes without disturbing the buffy coat layer. The plasma samples were centrifuged at 16000 g for 10 min, and then the supernatant was removed to a new tube without disturbing the pellet. The plasma was stored at −80°C until further analysis. cfDNA was recovered from 4 to 5 ml of the plasma samples using the QIAamp circulating nucleic acid kit according to the manufacturer's standard protocol (Qiagen). cfDNA was quantified using the Qubit 2.0 Fluorometer with dsDNA HS assay kits (Life Technologies). The starting material for subsequent testing consisted of 50 ng cfDNA prepared by the Qiagen method. DNA was profiled using a commercially available capture‐based sequencing panel. Indexed samples were subjected to capture‐based targeted sequencing using a panel consisting of 168 lung cancer‐related genes.²⁵, ²⁶ DNA was hybridized with the capture probe baits, selected with magnetic beads, and amplified by polymerase chain reaction (PCR). A bioanalyzer high sensitivity DNA assay was then used to assess the quality and size range, and 30 indexed samples were sequenced onto a NextSeq 500 (Illumina, Inc.) with pair‐end reads.