Method development for DNA methylation on a single cell level The - - PowerPoint PPT Presentation

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Method development for DNA methylation on a single cell level The - - PowerPoint PPT Presentation

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Method development for DNA methylation on a single cell level The project is carried out in TATAA Biocenter, Gothenburg, Sweden/ Secondment at Babrahaminstitute , Cambridge Dr. Bentolhoda Fereydouni, , Marie curie Postdoctoral research fellow

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Method development for DNA methylation on a single cell level

The project is carried out in TATAA Biocenter, Gothenburg, Sweden/ Secondment at Babrahaminstitute , Cambridge

  • Dr. Bentolhoda Fereydouni,

, Marie curie Postdoctoral research fellow Presented in final Epitrainmeeting , University College London, 16th

th January 2017

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Outline

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  • Introduction
  • Aims of the study
  • Reduced representation bisulfite sequencing

(RRBS)

  • Bismark software and data analysis
  • Summary & Discussion
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What is Epigenetics?

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From The Cell Biology of Stem Cells (2010)

1) histone modification 2) DNA methylation

  • Is the study of heritable changes in gene expression

(on or off the genes) that does not involve changes to the underlying DNA sequence

  • A change in phenotype without a change in genotype

Introduction

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Epigenetic inheritance as a form of Lamarckism

Lamarckism (or Lamarckian inheritance) is the idea that an organism can pass on characteristics that it has acquired during its lifetime to its offspring . Lamarckism has continued as studies in the field of epigenetics have highlighted the possible inheritance of behavioral traits acquired by the previous generation.

(1744–1829)

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Effects of environmental factors

Epigenetic marks play important roles in defining different cell types in the body and can be influenced by environmental and nutritional factors.

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DNA Methylation

Cytosine 5-methyl Cytosine DNA methyl-transferases DNA-demethylase(s)? TETs? Passive demethylation?

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  • symmetric

CG context

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Silencing of gene expression

Tissue differentiation and embryonic development

Faults in correct DNA methylation may result in

  • early development failure
  • epigenetic syndromes
  • cancer

Regula latio ion by DNA meth thyla latio ion

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DNA methylation is reset during reprogramming

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http://www.cell.com/cms/attachment/2002984389/2011335745/gr1.jpg

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Hypermethylation of CpGs leads to cancer

http://www.ks.uiuc.edu/Research/methylation/normal_cancer_cell.png

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Measuring DNA methylation by Bisulfite-sequencing

Image by Illumina

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Unmethylated cytosine produce uracil in DNA

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  • Establish a method to evaluate methylation profiles in single

cell material

  • Gel-free RRBS protocol and try to reduce the number of

purification steps

  • Using computational and statistical method for detecting and

analyzing covered CpG sites at the single cell level

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Aims of the Study

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Reduced representation bisulfite sequencing (RRBS)

  • is an efficient and high-throughput technique used to analyze the genome-

wide methylation profiles on a single nucleotide level.

  • This technique combines restriction enzymes and bisulfite sequencing in order

to enrich for the areas of the genome that have a high CpG content.

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Five steps in the standard RRBS method

1- Genomic DNA purification 2- Restriction enzyme digestion -cuts at CCGG sites. This enriches for CpG rich regions of the genome 3- End repair and dA tailing 4- Adapter ligation 5- Bisulfite conversion

All the five steps into a single-tube reaction

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Artificial methylation calls in RRBS libraries

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C genomic cytosine C unmethylated cytosine

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The final RRBS libraries were prepared with fragment analyzer and the concentration and size distribution were checked with Pico green/ Nanodrop.

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The Fragment Analyzer results of two RRBS libraries

Typical DNA size distribution in RRBS libraries ranges from 150 to 350 bp with visible peaks corresponding to MspI fragments 33 pg hgDNA 6.6 pg hgDNA

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The Fragment Analyzer results of two human tomour RRBS libraries

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The Fragment Analyzer results of two human Oocyte RRBS libraries

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BS-Seq Analysis Workflow

Sequencing Processing pipeline Methylation Analysis

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Explore and understand data

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Illumina platforms

HiSeq 2500 2000Mreads/run 400 Gb/run 2x100 2x125 bp Miseq 15-22Mreads/run 4,5-13,2 Gb/run 2x300 NextSeq500 400 Mreads/run 120 Gb/run 2x150 2x150

Production power Flexible power Focused power

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BS-Seq Analysis Workflow

QC Trimming Mapping Methylation extraction Mapped QC Analysis

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Taken from Babraham institute bioinformatics home page

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Data analysis for RRBS data

Bioinformatic analysis methods for DNA methylation profiling data with bioinformatic tools Bismark program adapter trimming with Trim Galore.

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What is Bismark?

  • Is a set of tools for the time-efficient analysis of Bisulfite-Seq (BS-Seq) data.
  • Performs alignments of bisulfite-treated reads to a reference genome and cytosine methylation calls

at the same time.

  • is written in Perl and is run from the command line.
  • Bisulfite-treated reads are mapped using the short read aligner Bowtie .
  • Bowtie (http://bowtie-bio.sourceforge.net/index.shtml) or Bowtie 2 http://bowtie-

bio.sourceforge.net/bowtie2 needs to be installed on computer.

Taken from Babraham institute bioinformatics home page

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Reference genome

First reference genome downloaded and place it in a genome folder. Genomes can be obtained from the Ensembl (http://www.ensembl.org/info/data/ftp/index.html/) orNCBI website (ftp://ftp.ncbi.nih.gov/genomes/)

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Bismark workflow

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Quality Control shows :

  • Number of sequences
  • Basecall qualities
  • Base composition
  • Potential contaminants
  • Expected duplication rate

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www.bioinformatics.babraham.ac.uk/projects/fastqc/

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QC: Base Composition

Oocytes Tumour cells

  • Typical BSSeq experiments in mammals tend to have an average cytosine content of ~1-2%

throughout the entire sequence length

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Duplication rate(RRBS has high duplication rate)

Oocytes Tumour cells

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Removing adapter contamination

before trimming after trimming

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Summary Adapter/Quality Trimming

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Important to trim because failure to do so might result in:

  • Low mapping efficiency
  • Mis-alignments
  • Errors in methylation calls since adapters are methylated
  • Basecall errors tend toward 50% (C:mC)
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Meth thyla latio ion bia ias

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good opportunity to look at conversion efficiency

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Bismark run report

  • Summary of alignment parameters used.
  • Number of sequences analyzed.
  • Number of sequences with a unique best alignment (mapping efficiency).
  • Statistics summarizing the bisulfite strand the unique best alignments came from.
  • Number of cytosines analyzed.
  • Number of methylated and unmethylated cytosines.
  • Percentage methylation of cytosines in CpG, CHG or CHH context
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Summary& Discussion

  • The advantage of the scRRBS method is, its applicability to subnanogram levels
  • f DNA as starting material, down to a single cell.
  • Particularly useful when the starting materials are very limited and precious,

such as mammalian early embryos and primordial germ cells.

  • Enables the heterogeneity of DNA methylomes among individual cells to be

studied, which may have important roles in biological processes such as cell differentiation, memory formation and oncogenesis.

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  • Since RRBS is highly sensitive, this technique can be used to quickly look at

aberrant methylation in cancer

  • If samples from the patient's tumor and normal cells can be obtained, a

comparison between these two cell types can be observed

  • A profile of the overall methylation can be produced quite rapidly
  • This technique can rapidly determine the overall methylation status of cancer

genomes which is cost and time effective

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Seminars and Presentations

  • 1. B. Fereydouni “Method development for DNA methylation on a single cell level” presented in EpiTrain Final
  • meeting. University College London, London, UK, 16th January 2017.
  • 2. B. Fereydouni, E.Hanson, A. Ståhlberg A and R. Sjöback “Gel-free reduced representation bisulfite

sequencing for single cell DNA methylation profiling” The Biology of Genomes meeting,10 – 14 May 2016, Cold Spring Harbor Laboratory, New York, USA, Poster presentation.

  • 3. B. Fereydouni “Method development for DNA methylation on a single cell level” presented in EpiTrain annual
  • meeting. Epigenomics of Common Diseases conference and EpiTrain annual meeting, Wellcome Genome Campus,

Hinxton, Cambridge, UK 6-9 November 2015.

  • 4. B. Fereydouni “Find and establish a method to evaluate methylation profiles in single cell material?” Novel

technologies in Epigenetics, Technology transfer and Entrepreneurship, EpiTrain annual meeting, IDIBELL, L’Hospitalet de Llobregat, Barcelona, Spain 15-16 October 2015.

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Courses

  • Multiplex PCR, 11th December 2015, TATAA Biocenter. Gothenburg, Sweden.
  • Sample preparation and quality control, 10th December 2015, TATAA Biocenter. Gothenburg, Sweden.
  • 2- Day NGS – Library construction and quality control, 26- 27th November 2015, TATAA Biocenter. Gothenburg, Sweden.
  • Methods and applications for microRNA analysis, 9th October 2015, TATAA Biocenter. Gothenburg, Sweden.

Genotyping with qPCR, 8th October 2015, TATAA Biocenter. Gothenburg, Sweden.

  • 2- Day Digital PCR- Application and analysis, 10-11th September 2015. TATAA Biocenter. Gothenburg, Sweden.
  • 3- Day Hands-on qPCR, 7-9th September 2015. TATAA Biocenter, Gothenburg, Sweden
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Thank you

  • Dr. Ellen Hanson
  • Dr. Robert Sjöback
  • Dr. Gavin Kelsey
  • Dr. Felix Krueger

All my colleagues at TATAA biocenter

Professor Ståhlberg

  • Dr. Kirsten Ward

Professor Mikael Kubista Professor Stephan Beck