sequencing analysis
The microbial DNA from fecal and BALF samples was extracted using a E.Z.N.A.Stool DNA Kit (Omega, Inc., USA), dissolved in Elution buffer and stored at -80 °C until detection. Nuclear-free water was used for blank. The V3-V4 region of the bacterial 16s rDNA gene was amplified in a total volume of 25 μL reaction mixture containing 25 ng of template DNA, 12.5 μL PCR Premix, 2.5 μL of each primer, and PCR-grade water to adjust the volume. The amplification conditions were 98 °C for 30 seconds, 32 cycles of denaturation at 98 °C for 10 seconds, annealing at 54 °C for 30 seconds, and extension at 72 °C for 45 seconds; and then final extension at 72 °C for 10 minutes. The primers used in this study were as follows: 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’).
The samples were sequenced using an Illumina NovaSeq platform provided by LC-Bio. In 16sDNA analysis, the Alpha- and Beta- diversity were calculated by QIIME2 and graphed by R package. Blast was used for sequence alignment, and the feature sequences were annotated with SILVA database for each representative sequence. Other diagrams were implemented using the R package (v3.5.2).