DNA Extractions and RRBS Data Analysis
Reduced Represented Bisulfite Sequencing (RRBS) Genomic DNA from F1 and
F2 liver were isolated using the Zymo Quick-DNA Mini Prep Kit (Zymo
Research Corp.). Upon submission, the gDNA samples were quantified using
a fluorometric PicoGreen assay and analyzed for quality using the
Nanodrop. All sampled passed the Input DNA Requirements outlines in
Tecan’s Ovation RRBS Methyl-Seq System (Publication Number:
M01394v7). gDNA samples were converted to sequencing libraries
using Tecan’s Ovation RRBS Methyl-Seq with TrueMethyl oxBS
System following manufacturers protocols (Part # 9522-A01). In summary,
extracted gDNA was digested by MspI and then ligated to indexed
sequencing adapters. Libraries were bisulfite converted (oxBS module was
not used) and amplified. Final library size distribution was validated
using capillary electrophoresis and quantified using fluorimetry
(PicoGreen). Libraries were pooled and sequenced on one lane of an S4
2x150 paired-end run on the Illumina NovaSeq 6000 System.
R (R Core Team (2021). R: A language and environment for statistical
computing. R Foundation for Statistical Computing, Vienna, Austria.
https://www.R-project.org/.) and Linux command line tools were
used for RRBS analysis. FastQC (version11.3) assessed overall sequencing
quality, and TrimGalore(F. Krueger 2015) (version 4.5) with default
settings trimmed low-quality bases, adapter sequences, and end-repair
bases from 3’ ends. Bismark(Felix Krueger and Andrews 2011) (version
19.0) aligned and called methylation. Reads were aligned to mm10 mouse
reference genome using Bowtie2(Langmead and Salzberg 2012) (version
2.3.4) using default parameters. Methylation calls were reported for all
nucleotides with the minimum read depth of 5. Paired raw fastq.gz reads
were uploaded to the Sequence Read Arcive and can be accesed under
Bioproject Number (PRJNA881737) . The DSS package in R (version
2.32.0) detected differential methylation in liver for F1 and F2 females
versus sex- and generation- matched 1ppb controls (n = 3 per group) for
sites with a minimum coverage of 10 reads. The DMLtest and callDML
functions were used to detect differentially methylated CpGs (DMCs).
Smoothing was set to FALSE in DMLtest as is recommended for sparse data.
Differentially methylated regions (DMRs) were identified with callDMR.
The p-value threshold was set to FDR < 0.001 for both tests
with delta > 0.01 required to call DMCs. A delta value was
not enforced for DMRs; all CpGs within a potential DMR need not exhibit
the same direction of change, meaning a delta threshold could lead to
false negatives. The Annotatr R package was used to annotate DMCs and
DMRs to CpG islands, genes, and intergenic regions in the mm10
genome(Cavalcante and Sartor 2016). The functions plot_annotation and
plot_categorical functions were used to generate figures.