title: RNA Seq Mouse: Stool S28R1R2
design: Libraries were prepared using the 16S metagenomic sequencing library preparation (Illumina workflow). In this workflow, 16S amplicons were generated using KAPA HiFi HotStart ReadyMix and primers specific to the 16S (V3-V4) region (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'; 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3'). Purified amplicons were indexed with Illumina Nextera XT Index Primers and purified with Ampure XP beads. 16S libraries were pooled in equal amounts based on fluorescence quantification. Final library pools were quantitated by quantitative PCR and sequenced on an Illumina MiSeq using MiSeq v3 600 cycle chemistry. For data analysis, paired-end reads were assigned to samples based on their unique barcodes. After trimming of primer sequences by cutadapt (v1.9), sequencing reads were merged into tags based on overlapping regions using FLASH (v1.2.8). Quality filtering was performed by fqtrim (v0.94) with sliding windows to retain sequences having average quality scores  20 and longer than 100 bp in size. Chimeric sequences were discarded using Vsearch (v2.3.4). After dereplication using DADA2 50, ASV feature sequences were obtained and quantified. Singleton ASVs were excluded from further analysis. Species annotations of ASVs were performed by SILVA (release 132, https://www.arb- silva.de/documentation/release138/) 51 and NT-16S. Alpha diversity and beta diversity were calculated based on relative abundance of ASVs 52. QIIME2 was used for alpha diversity analyses, including Chao1, Observed_species, Goods_coverage, Shannon, and Simpson indices. Beta diversity analysis was similarly performed by QIIME2. Graphs were generated by ggplot2 (R package v3.5.2). Differential analysis was performed based relative abundance values. Mann-Whitney U test was used for comparisons between 2 groups. Kruskal-Wallis test was used for comparisons among 3 or more groups. A species with p< 0.05 was considered significantly differential species. Bioinformatics services and analyses including diversity, taxonomy, and differential analyses for bacterial families were provided by LC Sciences (Houston, Texas).