Metagenomic Analysis of Respiratory Specimens

First, DNA and RNA were extracted using Magnetic serum/plasma DNA Maxi kit (Tiangen Biotech (Beijing) Co. Ltd., China). For BALF specimens, HostZERQ Microbial DNA kit (Jianshi Biotech (Beijing) Co. Ltd., China) was used to remove human nucleic acids for further nucleic acid extraction. Second, nucleic acids were fragmented into 150–300 bp in length with Bioruptor (Diagenode Diagnostics, Belgium) that is a non-contact ultrasonic disruptor. The library was constructed using Library Preparation kit (Kapabio System, Boston, MA). Third, high-throughput sequencing was conducted with Illumina Next Seq550Dx system (Illumina Inc., San Diego, CA). In the process of sequencing, adaptors, reads with low quality and repeated sequences, and short reads < 36 bp in length were removed. Microorganisms were then identified in the specimens through sequence alignment in the microbial genome database (bacteria, viruses, fungi and parasites) by using Bowtie2 (version 2.3.5) (Genoxor Medical Science and Technology Inc., Shanghai) [24 (link)].