Lung microbiota analyses in asthma mouse model

Analysis of microbiota composition. Bacterial DNA was extracted from BAL pellets according to the manufacturer’s instructions (Macherey Nagel, genomic DNA from tissue, Nucleospin Tissue). The microbiota composition was analyzed by 16S sequencing with permission from Biofortis (Saint-Herblain, France). Raw sequencing data were obtained from a single Illumina MiSeq run as 250 bp paired-end reads targeting the V3-V4 region (Primers : Bakt_341F 5′-CCTACGGGNGGCWGCAG-3′,Bakt_805R 5′ GACTACHVGGGTATCTAATCC-3) of the 16S rDNA gene. Reads were processed with microSysMics (https://bio.tools/microSysMics), a workflow built around the QIIME2 toolbox, chaining softwares in order to automatize metabarcoding analysis. PCR primers and remaining Illumina adapters were removed with Cutadapt. ASVs inference and count estimation were performed with dada2 using a trimming length of 220 and default parameters. We used a Naive Bayes Classifier pre-trained on the SILVA 99% reference database (Release 138) to assign ASVs to taxa.