Extended Data Fig. 11: DNA methylation and small RNA profiles in purified sperm from dysbiotic males.
From: Paternal microbiome perturbations impact offspring fitness
(a) Boxplot showing the global level of DNA methylation in sperm from multiple independent males, (n = 5 per condition) ascertained by whole-genome bisulfite sequencing (WGBS), quantified using 50 CpG tiles. Bar represents median and whiskers indicate median plus/minus the IQR multipled by 2. (b) Example genome track showing highly reproducible DNA methylation patterns in sperm irrespective of nABX treatment. Each data point represents the percentage methylation across a 50 CpG tile. Grey regions demarcate hypermethylated genomic zones. (c) Volcano plot highlighting significant differential methylated regions (DMR). Whilst 21 DMR loci were identified, the effect size was modest and they primarily overlapped CpG shores; loci more prone to natural biological or technical variation. (d) Genome track showing DNA methylation changes at a representative DMR (in green) using read-depth sensitive logistic regression (p < 0.05 & >20% abs. change). Percentages indicate DNA methylation level across the DMR in each sperm sample. Data points indicate methylation at each sliding 50 CpG tile. (e) Scatter plot showing DNA methylation at genomic imprinted regions in control and nABX-treated sperm. Paternal and maternal imprints are indicated. (f) Bar chart showing small RNAs that exhibit a foldchange in expression in purified sperm from nABX males relative to control sperm. (g) Absolute abundance of each indicated small RNA and class in control or nABX sperm. tRNA-Gly-GCC-2 (and tRNA-Gly-GCC-4) represent a major constituent of total RNA abundance in mature sperm and therefore relatively small foldchange differences in their expression constitutes a major change in absolute copy number in sperm heads. Each sample represents a total of n = 9 individual male samples pooled in to three replicates. Inset is the overall abundance (proportion of reads) of each small RNA class (h) Zoomed in representation of (B) showing miR-141 and Let-7 are highly abundant among microRNA and piRNA classes. (i) TaqMan qPCR quantification of top hits (miR-141-3p and tRNA-Gly-GCC) from small RNA-seq datasets using purified sperm from nABX-exposed males. Bars show mean with S.D and significance by one-tailed t-test. Sperm samples obtained in independent treatments from samples used for small RNA-seq (CON n = 4; nABX n = 4).
