DNA-Seq of BALF
Experienced clinicians guided the bronchoscopy to diseased region of the lungs referring to CT results. Sterile normal saline was instilled and then retrieved by gentle suction as BALF sample. Centrifuge the BALF samples at 1000 rpm for 10 min at 4 C and supernatants and precipitation were separately frozen (80 C) for further analysis. DNA was extracted from the precipitation using a QIAamp UCP Pathogen DNA Kit (Qiagen) following the manufacturer provided protocol. Human DNA was removed using Benzonase (Qiagen) and Tween20 (Sigma).10 nanograms DNA samples was used to construct library using Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA). Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate approximately 20 million reads for each library. Peripheral blood mononuclear cell samples with 105 cells/mL from healthy donors was served as negative controls. DNA-free water also went through DNA extraction and mNGS analysis as a blank control group to assess the degree of background contamination.