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.