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Reduced Representation Bisulfite Sequencing (RRBS-Gene Methylation)

Overview

DNA methylation research has always been a hot topic in disease research and is closely related to gene expression and phenotypic traits. RRBS is an accurate, efficient and economical method for DNA methylation research. Enrichment of promoter and CpG island regions by enzymatic cleavage (Msp I), combined with Bisulfite sequencing, provides high resolution DNA methylation detection.

Applications

Applications range from transcriptional regulation to developmental pathways to disease mechanisms and beyond.

For medical research:

  • Pathological mechanism
  • Tumor-subtypes classification
  • Molecular markers
  • Human evolution
  • Drug target

For agricultural research:

  • Development
  • Adaptability
  • Agronomic traits

Advantages

  • Unsurpassed data quality: We guarantee that ≥ 80% of bases have a sequencing quality score ≥ Q30, exceeding Illumina’s official guarantee of ≥ 75%.
  • Comprehensive data analysis: We use industry standard software such as Bismark and a mature in-house pipeline for mapping, Differentially Methylated Region (DMR) analysis, functional analysis and data visualization.

Sample Requirements

 

 

Sample Type Required amount Purity
Genomic DNA ≥ 1.5 μg OD260/280=1.8-2.0

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Sequencing Parameter and Analysis

Platform Type Illumina Novaseq 6000
Read Length Pair-end 150
Recommended Sequencing Depth ≥ 10Gb clean data per sample
Standard Data Analysis
  • Data quality control
  • Alignment to reference genome
  • Quality controls for 5-mC identification
  • mCs detection, methylation level calculation
  • Methylation level and frequency distribution in different structural sequences
  • Differentially methylated regions (DMRs), Differentially Methylated Promoter (DMPs) detection and annotation
  • Function enrichment of DMR-associated genes and DMP-associated genes
  • Visualization of BS seq data
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    Note: Sequencing depths and bioinformatic analysis requests can be customized based on the project needs. Please contact us for more information.

    Project Workflow

    Sample Quality Control

    Library Quality Control

    Data Quality Control

    Sample Preparation

    Map / Digestion

    Library Construction with Bisulfite Treatment

    Sequencing

    Bioinformatics Analysis

    Association of a History of Child Abuse with Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence

    Background:

    Child abuse has devastating and long-lasting consequences, considerably increasing the lifetime risk of negative mental health outcomes such as depression and suicide. In this study, RRBS and RNA-seq were used to explore the underlying mechanisms that increased the risk of mental health disease caused by childhood abuse. The results showed that a history of child abuse was associated with cell type‒specific changes in DNA methylation of oligodendrocyte genes and a global impairment of the myelin-related transcriptional program. These effects were absent in the depressed suicide completers with no history of child abuse, and they were strongly correlated with myelin gene expression changes observed in the animal model.

    Sampling & Sequencing Strategy:

    1. Samples:
    Postmortem brain samples from:
    • depressed individuals who died by suicide with a history of severe child abuse, N=27
    • depressed individuals who died by suicide without a history of severe child abuse, N=25
    • psychiatrically healthy control subjects, N=26

    2. Library preparation:
    RRBS library and RNA-seq library

    3. Sequencing:
    Illumina platform, PE100 and SE50

    4. Bioinformatics analysis:
    Differential methylation analysis (RRBS) and Differential expression analysis (RNA sequencing)

    Results & Conclusion:

    Epigenetic Effects of Child Abuse

    Figure 1. Epigenetic adaptations in the anterior cingulate cortex following child abuse.

    1) Widespread differences of DNA methylation were uncovered between child abuse and control group.
    Both hyper- and hypomethylation were detected in the child abuse group compared with the control group, suggesting that child abuse bidirectionally regulates epigenetic patterns in the cingulate cortex. The three most significantly differentially methylated regions intersected with genes directly related to myelin and oligodendrocytes: LINGO3, POU3F1 and ITGB1 (Fig. 1).

    Figure 2. DNA methylation was measured in sorted populations of NeuN+ and Sox10+ nuclei, focusing on differentially methylated loci identified by RRBS.

    2) DNA methylation patterns are cell type specific
    For both LINGO3 and POU3F1 decreased methylation in the child abuse group was confirmed and found to occur specifically in oligodendrocyte, but not neuronal, nuclei. These effects were absent in the depressed group. However, no significant DNA methylation differences for ITGB1 in either Sox10+ or NeuN+ nuclei between groups (Fig. 2).

    Reference: Lutz P, Tanti A, Gasecka A, et al. A ssociation of a History of Child Abuse with Impaired Myelination in the Anterior Cingulate Cortex: Convergent Epigenetic, Transcriptional, and Morphological Evidence[J]. American Journal of Psychiatry, 2017, 174(12): 1185-1194.


    Figure 1 Distribution of genome coverage.png


    Figure 2 Methylation level distribution in whole genome.png


    Figure 3 Circos plots for methylation density on chromosome.png


    Figure 4 Heatmap analysis of gene functional region methylation levels-1.png


    Figure 5 An overview of methylation level distribution at functional genetic elements.png


    Figure 6 KEGG enrichment scatter.png