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.