2.6 Microbiota data generation and processing
After DNA extraction, the targeted gene for bacterial taxonomic affiliation using broad bacterial primers of the region V4 of the 16S rRNA gene (515F‐806R, 390 bp) was amplified through PCRs. Amplification, library preparation and sequencing were carried out in a similar manner to MHC genotyping, with a depth of 100,000 reads/samples of two libraries composed of 48-52 samples.
The quality controls of the demultiplexed paired‐end reads were performed through the software FastQC (Andrews, 2010). Demultiplexed sequence reads denoising and amplicon sequence variants (ASVs) picking steps were done with the QIIME2 tool (Bolyen et al., 2018; v. 2019.1), using the DADA2 pipeline (Callahan et al., 2016; Callahan et al., 2017). Samples were pooled by individuals to limit bias from diet foods prior to rarefaction. Rarefaction was conducted at 27,000 reads/samples in sampling depth. ASVs—or also referred to as bacterial phylotypes—were then screened to the 97% 16S rRNA gene full‐length reference sequences from the Silva v.132 database (Pruesse et al., 2007) for taxonomical association using the VSEARCH classifier implemented in QIIME2 (Bokulich et al., 2018). Sequence alignment and phylogeny building were conducted in QIIME2 for the construction of UniFrac distance matrices.