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).