title: 16s rRNA gene sequence analysis of gut microbiome from rats treated with aflatoxin B1
design: Library construction:   A two-step Quadruple-index PCR method was used to prepare the 16s rRNA gene libraries. Briefly, locus-specific primers to the V3 and V4 regions of 16s rRNA (Klindworth et al., 2013): S-D-Bact-0341-b-S-17 (Bakt_341F) CCTACGGGNGGCWGCAG and S-D-Bact-0785-a-A-21 (Bakt_805R) GACTACHVGGGTATCTAATCC) were fused with Illumina TruSeq Read1 and Read2 sequences.  Internal tag sequences 5 bases long (Faircloth and Glenn, 2012) with 0-3 base spacers were inserted between the Illumina and locus-specific portions of the oligonucleotides.    Following generation of the primary amplicons, another pair of fusion oligonucleotides was used as primers to generate full-length dual-indexed Illumina TruSeqHT style libraries. The resultant amplicons were normalized, purified 1:1 with Speedbeads (Faircloth and Glenn, 2014). Sequencing of these 16s rRNA fragment libraries was performed in the Georgia Genomic Facility (University of Georgia, Athens, GA) using the Illumina MiSeq with v2 500 cycle chemistry, resulting in paired-end 250 base reads to obtain ~30,000 reads per sample.  Experiment Design:  12 male Fischer 344 rats (5 weeks old, 120-130g) were acclimatized for 1-week before the experiment, randomly allocated into four dosing group: control, low (5 μg AFB1/kg body weight), medium (25 μg/kg), high (75 μg/kg); upon starting of the experiment, gavaged with control vehicle (DMSO) or AFB1 in DMSO (< 50 µL/animal), 5 days per week for a duration of 4 weeks. Rat feces were collected at the end of the fourth week in metabolic cages and stored at -80°C.