Topic outline

  • General

    DNA methylation and methylome analysis with NGS

    Fribourg, 13-16 February 2017

    This page is addressed to registered participants. To access course description and application form, please click here.

    For any assistance, please contact training@sib.swiss.

    Overview

    Methylations of the DNA are important modifications that affect the DNA of bacteria and higher eucaryotes. The DNA can be modified on different bases (6mA, 4mC, 5mC) and these modifications have various effects, e.g., on gene expression or protection against foreign DNA. These modifications can be transmitted from generation to generation, they are called epigenetics modifications. Current next generation sequencing technologies allow for the detection of those modifications at the genome level introducing the « methylome ». In addition Histone modifications are also part of the epigenomics modifications that can influence gene expression. These modification can be analyzed by NGS methods. In this workshop we will study the methods used to produce and to analyze this NGS data.

    Objectives

    Learn about DNA methylation and other chromatin modifications.

    Learn the tools and databases available to study the methylome.

    Requirements

    Requirements: a portable computer with WIFI and 4Gb RAM. Basic knowledge of the R package.

    Location

    • Feb 14: DNA methylation in Prokaryotes

      9:15 Theoretical part (60-90 min):

      intro Prokaryotic epigenetics
      - differences to eukaryotes: more diverse modifications, defined target sequence motifs, RM-systems, ...
      - SMRT sequencing technology, data analysis workflow,
      - brief review of recent literature: applications of SMRT sequencing & findings on biology

      10:30 Coffee break

      11:00 practical session (RStudio, web server)

      - What is the positional resolution of SMRT sequencing, determination of modified bases within specific target motif sequences?
      - How sensitive is SMRT sequencing, compute the fractions of motif instances with modifications detectable by SMRT in bacterial genomes?
      - Are the detected modifications associated with positions of specific genomic features?

      12:30 Lunch Break

      13:30 practical session 2 

      15:00 Coffee break

      15:30 practical session 2 

      • Feb 15: DNA methylation in Eukaryotes

        In this session, we will discuss DNA methylation in eukaryotes, with a focus on mammalian 5mC methylation of cytosines in CpG context.

        9:15 The first part (theory, around 1 hour) will present the topics:

        • Introduction and  role of DNA-methylation in mammals
        • How is DNA-methylation experimentally measured (technology)
        • Typical methylation patterns found in mammals (PMDs, LMRs, UMRs/CGI) and their biological interpretation
        • Outline of a typical analysis workflow and tools

        10:30 Coffee break

        11:00 Practical session

        In the second part (exercises, rest of the session), we will use R/Bioconductor on small sample datasets to:

        • go from raw bis-seq reads to cytosine methylation values
        • identify hypomethylated regions (methylome segmentation)
        • visualize and interpret the results

        For the second part (exercises) please install the required R packages as shown in the exercises handout in section 1 "Install required software".

        12:30 Lunch break

        13:30 practical session 2 

        15:00 Coffee break

        15:30 practical session 2 

        • Feb 16: Other chromatin epigenetic modifications (histones, etc.)

          9:15 Theoretical part (60-90 min)

          Principles of ChIP-Seq data analysis: a practical approach. Focus will be given to bioinformatics pipeline for quality controls, peak detection, annotation and functional analysis.

          10:30 Coffee break

          11:00 Practical (RStudio and terminal, in Linux Virtual Machine)

          Run QC tools (deepTools, ChIPQC), apply peak calling (MACS2), annotate the peaks (ChIPseeker), plot peak profiles and TSS heatmaps (ChIPseeker), differential binding analysis with DiffBind. Finally add the RNA-seq analysis information.

          12:30 Lunch break

          13:30 practical session 2 

          15:00 Coffee break

          15:30 practical session 2