sequences of bacteria
Microbiome bioinformatics were performed with QIIME 2 2019.4 (Bolyen et al. 2018) with slight modification according to the official tutorials (https://docs.qiime2.org/2019.4/tutorials/). Briefly, raw sequence data were demultiplexed using the demux plugin following by primers cutting with cutadapt plugin (Martin, M., 2011). Sequences were then quality filtered, denoised, merged and chimera removed using the DADA2 plugin (Callahan et al. 2016). Non-singleton amplicon sequence variants (ASVs) were aligned with mafft (Katoh et al. 2002) and used to construct a phylogeny with fasttree2 (Price et al. 2010). Alpha-diversity metrics (Chao1 (Chao, 1984), Observed species, Shannon (Shannon, 1948a, b), Simpson (Simpson, 1949), Faiths PD (Faith, 1992), Pielous evenness (Pielou, 1966) and Goods coverage (Good, 1953)), beta diversity metrics (weighted UniFrac (Lozupone et al. 2007), unweighted UniFrac (Lozupone et al. 2005), Jaccard distance, and Bray-Curtis dissimilarity) were estimated using the diversity plugin with samples were rarefied to XXX sequences per sample. Taxonomy was assigned to ASVs using the classify-sklearn naive Bayes taxonomy classifier in feature-classifier plugin (Bokulich et al. 2018a) against the Greengenes 13_8 99% OTUs reference sequences (McDonald et al. 2012).