Computational protocol: Enhanced offspring predisposition to steatohepatitis with maternal high-fat diet is associated with epigenetic and microbiome alterations

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Protocol publication

[…] DNA methylation changes associated with maternal HFD and offspring MCD challenge were assessed using RRBS, which involves sequencing of bisulfite-converted MspI fragment libraries. Liver genomic DNA was isolated using a combination of proteinase-K digestion and Purelink genomic DNA isolation kits (Life Technologies). Three biologically distinct pools of genomic DNA (containing n = 3 in each pool) were utilized to generate libraries. RRBS libraries were prepared as described by Gu et al [] with detailed methods provided in Supplementary methods. Reads were trimmed for adapter sequences using Trim Galore and filtered for quality score. Alignment and methylation calling were performed using Bismark. We first examined whether maternal HFD and offspring diets affected methylation of promoters and CpG islands (CGI). Promoters were sub-classified into those overlapping or devoid of CGIs. Frequency distribution of methylation status of features was computed. Statistical differences between groups were analyzed using χ2 test. Data analysis and summarization were done using SeqMonk and the DSS package in R []. Comparisons between different groups (CC, C-MCD, HC and H-MCD) were performed using Wald test, and P values were adjusted for multiple testing using the FDR method. CpGs with p < 0.0001 and a minimum difference in methylation (Δme) of 10% were retained. Differentially methylated regions (DMRs) were annotated with the closest/overlapping transcription start sites (TSSs) (±10 kb). The lists of differentially expressed genes were analyzed for GO biological process and molecular function enrichment using the BiNGO plugin in Cytoscape. [...] Bacterial DNA was isolated from cecal contents using QIAamp Fast DNA stool mini kit (Qiagen) including a bead-beating step. Fifty nanograms of genomic DNA was utilized for amplification of the V4 variable region of the 16S rRNA gene using 515F/806R primers. Forward and reverse primers were barcoded to accommodate multiplexing up to 384 samples per run as described by Kozich et al []. Paired-end sequencing (2 X 250 bp) of pooled amplicons was carried out using Illumina Miseq platform with ~30% PhiX DNA.Processing and quality filtering of reads was performed by using scripts in QIIME (v1.9.1) []. OTU picking was performed using an open-reference method and representative sequences were further aligned using PyNAST with the Greengenes core-set alignment template. Construction of the phylogenetic tree was performed using QIIME. Alpha rarefaction was performed using the phylogenetic diversity, Chao1 and observed species metrics. β-diversity estimation was carried out by computing weighted and un-weighted UniFrac distances between samples using QIIME. Differences in OTU abundance between groups were identified using STAMP [] and visualized using Clustvis. PICRUSt was used to identify differences in predictive metagenome function []. We also examined group difference using LefSe which utilizes Linear Discriminant Analysis of Effect Size. Associations of OTU abundance with serum ALT levels were performed using MaAsLin which is a multivariate statistical framework that performs boosted, additive general linear models. Analysis for LefSe and MaAsLin were carried out using the default settings []. […]

Pipeline specifications

Software tools Trim Galore!, SeqMonk, BiNGO
Application BS-seq analysis
Diseases Choline Deficiency, Fatty Liver, Liver Diseases, Neoplasms, Adipose Tissue, Non-alcoholic Fatty Liver Disease