Advancing Epigenetics Towards Systems Biology

Bioinformatics

Pedro Madrigal1,2

Introduction

Precisely mapping protein-DNA binding to genomic sites is a pivotal task in order to understand gene regulation. Chromatin immunoprecipitation (ChIP) followed by microarray hybridization (ChIP-chip) or sequencing (ChIP-seq) have been extensively used to map transcription factor binding sites (TFBSs), with ChIP-seq comparing favourably with respect to ChIP-chip in terms of resolution and signal-to-noise ratio (Ho et al., 2011). While ChIP-seq remains the standard, most-used methodology (Furey, 2012), λ exonuclease digestion followed by high-throughput sequencing, or ChIP-exo, has recently emerged as a powerful and promising technique able to substitute ChIP-seq, and to circumvent its limitations (Rhee and Pugh, 2011; Mendenhall and Bernstein, 2012). In this protocol, the distribution of mapped reads is characterised by pairs of two distinct peaks, one at each DNA strand, centred at the λ exonuclease borders and separated frequently at fixed distances (Rhee and Pugh, 2011). Importantly, the improved resolution of ChIP-exo can provide novel insights into protein-DNA interactions (Rhee and Pugh, 2011; Serandour et al., 2013). Furthermore, ChIP-exo distinguishes weaker peaks more confidently, and also closely-located binding events, that in ChIP-seq are generally unresolved or deconvolved through computational approaches (e.g., Guo et al. (2012)). [...]

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Pedro Madrigal1,2

1 Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
2 Wellcome Trust-MRC Cambridge Stem Cell Institute, Anne McLaren Laboratory for Regenerative Medicine, Department of Surgery, University of Cambridge, Cambridge, CB2 0SZ, UK

Corresponding author: Pedro Madrigal
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Pedro Madrigal

Nicolas Delhomme1, Niklas Mähler2, Bastian Schiffthaler1, David Sundell1, Chanaka Mannapperuma1, Torgeir R. Hvidsten1,2, Nathaniel R. Street1,3

Introduction

RNA-Seq (RNA-Sequencing) has fast become the preferred method for measuring gene expression, providing an accurate proxy for absolute quantitation of messenger RNA (mRNA) levels within a sample (Mortazavi et al, 2008). RNA-Seq has reached rapid maturity in data handling, QC (Quality Control) and downstream statistical analysis methods, taking substantial benefit from the extensive body of literature developed on the analysis of microarray technologies and their application to measuring gene expression. Although analysis of RNA-Seq remains more challenging than for microarray data, the field has now advanced to the point where it is possible to define mature pipelines and guidelines for such analyses. However, with the exception of commercial software options such as the CLCbio CLC Genomics Workbench, for example, we are not aware of any fully integrated open-source pipelines for performing these pre-processing steps. Both the technology behind RNA-Seq and the associated analysis methods continue to evolve at a rapid pace, and not all the properties of the data are yet fully understood. Hence, the steps and available software tools that could be used in such a pipeline have changed rapidly in recent years and it is only recently that it has become possible to propose a de-facto standard pipeline. Although proposing such a skeleton pipeline is now feasible there remain a number of caveats to be kept in mind in order to produce biologically and statistically sound results. [...]

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Nicolas Delhomme1, Niklas Mähler2, Bastian Schiffthaler1, David Sundell1, Chanaka Mannapperuma1, Torgeir R. Hvidsten1,2, Nathaniel R. Street1,3

1 Umeå Plant Science Center, Department of Plant Physiology, Umeå University, 90187, Umeå, Sweden
2 Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, 1432 Ås, Norway
3 Computational Life Science Cluster (CLiC), Umeå University, Umeå, Sweden

Corresponding author: Nicolas Delhomme
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Nicolas Delhomme, Niklas Mähler, Bastian Schiffthaler, David Sundell, Chanaka Mannapperuma, Torgeir R. Hvidsten<sup>1,2</sup>, Nathaniel R. Street<sup>1,3</sup>

Felix Krueger, Simon R Andrews

Introduction

Dramatic improvements and falling costs of high throughput sequencing have made bisulfite sequencing (BS-Seq) a viable option for the global analysis of DNA methylation (Bock et al, 2011; Li et al, 2010; Lister et al, 2009; Lister et al, 2011; Meissner et al, 2008; Stadler et al, 2011; Xie et al, 2012). The analysis of methylation obtained from BS-Seq is relatively straight forward, but care should be taken for initial quality control, trimming and suitable alignment of BS-Seq libraries since these are susceptible to a variety of errors or biases that one could probably get away with in other sequencing applications (discussed in (Krueger et al, 2012)). [...]

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Felix Krueger, Simon R Andrews

Bioinformatics Group, The Babraham Institute, Cambridge, CB22 3AT, United Kingdom

Corresponding author: Felix Krueger & Simon R Andrews
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Felix Krueger, Simon R Andrews

Ruhi Ali1, Florence M.G. Cavalli1, Juan M. Vaquerizas1, Nicholas M. Luscombe2,3

Introduction

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is becoming the standard experimental procedure to investigate transcriptional regulation and epigenetic mechanisms on a genome-wide scale (reviewed in (Park, 2009)). The technique involves covalent cross-linking of proteins to the DNA, followed by fragmentation and immunoprecipitation (IP) of the chromatin by using an antibody against the protein or histone modification of interest. The result of this experiment is a set of short DNA fragments of about 200 bp in length that represent regions of the genome where the protein is bound, or where specific histone modifications occurred. The segments are then sequenced using one of the various next generation sequencing procedures now available. The resulting reads (usually 36 to 100bp) are then mapped back to the reference genome of interest in order to identify regions with significant binding [...]

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Ruhi Ali1, Florence M.G. Cavalli1, Juan M. Vaquerizas1, Nicholas M. Luscombe2,3

1 European Bioinformatics Institute. Wellcome Trust Genome Campus, Cambridge CB10 1SD, UK.
2 Okinawa Institute of Science & Technology, 1919-1 Tancha, Onna-son, Kunigami- gun, Okinawa 904-0495, Japan.
3 University College London Genetics Institute, Gower Street, London WC1E 6BT, UK

Corresponding author: Nicholas M. Luscombe
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Ruhi Ali, Florence M.G. Cavalli, Juan M. Vaquerizas, Nicholas M. Luscombe

Matthias Siebert, Michael Lidschreiber, Holger Hartmann, and Johannes Söding

Introduction

Chromatin immunoprecipitation coupled to tiling microarray analysis (ChIP-on-chip) is used to measure genome-wide the DNA binding sites of a protein of interest. In ChIP-on-chip, proteins are covalently cross-linked to the DNA by formaldehyde, cells are lysed, the chromatin is immunoprecipitated with an antibody to the protein of interest and the fragmented DNA that is directly or indirectly bound to the protein is analyzed with tiling arrays. For this purpose, the fragmented DNA is fluorescently labeled and hybridized to the tiling array, which consists of millions of short (25 to 60 nucleotides long) probes that cover the genome at a constant spacing (4 to 100s of nucleotides), like tiles covering a roof. The data generated by one experiment consists of an intensity value for each DNA probe. These values measure the relative quantity of DNA at the probe's genomic position in the immunoprecipitated material. [...]

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Matthias Siebert, Michael Lidschreiber, Holger Hartmann, and Johannes Söding

Gene Center Munich - Ludwig-Maximilians-Universität - Feodor-Lynen-Str. 25 - 81377 Munich, Germany

Matthias Siebert, Michael Lidschreiber, Holger Hartmann, and Johannes Söding