2.4 mNGS
Sample Processing and Nucleic Acid Extraction : Lung biopsy specimen was collected and cut into small pieces. Samples of 0.5–3 ml BALF and soaking solution of brush tips were collected from patients following standard procedures, respectively. DNA was extracted using the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH) according to the manufacturer’s recommendation. Construction of DNA libraries : Single-stranded DNA circle (ssDNA circle) library was constructed after DNA-fragmentation, end-repair, adapter-ligation, DNA denaturation into single strands, DNA circularization. DNA nanoballs (DNBs) were generated from the ssDNA circle using rolling circle amplification (RCA). Finally, qualified DNBs were loaded on the flow cell and sequenced on BGISEQ-50 platform. Sequencing and bioinformatic analysis : High-quality sequencing data were generated by removing low-quality, and short (length < 35bp) reads, followed by a computational substraction of human host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment. After removal of low-complexity reads, the remaining data were classified aligning to four Microbial Genome Databases simultaneously, consisting of viruses, bacteria, fungi, and parasites. The databases were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). It contains 4,061 viral taxa whole genome sequence, 2,473 bacterial genomes or scaffolds, 199 fungi related to human infection, and 135 parasites associated with human diseases.