Improvement of the ChIP-seq technique for histone posttranslational modifications through enhanced laboratory protocols and data processing methods

Massively parallel sequencing has rapidly become the dominant technique in various omics studies as it provides an unequalled amount of high quality quantitative data for a reasonable cost that is decreasing ever since the first next generation sequencers appeared. Subsequently, chromatin immunoprecipitation coupled with massively parallel sequencing (ChIP-seq) is the prevailing method of choice for investigating protein–DNA interactions in a genome-wide manner. Bioinformatics tools are also evolving quickly to meet the increasing demands of processing huge amounts of ChIP-seq data and to open the way for novel techniques and insights. However areas still exist that could benefit from improved wet-lab and dry-lab methods. One such area is data visualisation and interpretation; another is the ChIP-seq study of histone posttranslational modifications, especially the research of inactive histone marks which tend to produce diffuse broad enrichments instead of point-source peaks. Achieving proper enrichment and unbiased analysis in such histone mark studies proves to be a great challenge. In this doctoral thesis we show how we addressed these issues on both the level of bioinformatics and the level of sample processing methods. We present our innovative analysis tools we developed to this end, among others a highly customisable, feature rich viewer for next generation sequencing data visualisation, and an analysis pipeline specifically aimed to handle broad enrichments from ChIP-seq studies of (inactive) histone marks. We propose specific software and specific peak calling settings to detect a range of histone modifications accurately, and we describe the way to determine the optimal settings. Along the pipeline we also present a protocol designed to enhance enrichments and facilitate peak detection in broad peak studies typical of inactive histone marks. We demonstrate how this method affects various enrichment types and propose potential applications that could benefit from it. Furthermore we show diverse achievements with the analysis pipeline, including the interpretation of the aforementioned wet-lab method, and the development of an automated ChIP-seq protocol optimised for low cell numbers

Automating ChIP-seq Experiments to Generate Epigenetic Profiles on 10,000 HeLa Cells

Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) is a technique of choice for studying protein-DNA interactions. ChIP-seq has been used for mapping protein-DNA interactions and allocating histones modifications. The procedure is tedious and time consuming, and one of the major limitations is the requirement for high amounts of starting material, usually millions of cells. Automation of chromatin immunoprecipitation assays is possible when the procedure is based on the use of magnetic beads. Successful automated protocols of chromatin immunoprecipitation and library preparation have been specifically designed on a commercially available robotic liquid handling system dedicated mainly to automate epigenetic assays. First, validation of automated ChIP-seq assays using antibodies directed against various histone modifications was shown, followed by optimization of the automated protocols to perform chromatin immunoprecipitation and library preparation starting with low cell numbers. The goal of these experiments is to provide a valuable tool for future epigenetic analysis of specific cell types, sub-populations, and biopsy samples

Geno viewer, a SAM/BAM viewer tool

The ever evolving Next Generation Sequencing technology is calling for new and innovative ways of data processing and visualization. Following a detailed survey of the current needs of researchers and service providers, the authors have developed GenoViewer: a highly user-friendly, easy-to-operate SAM/BAM viewer and aligner tool. GenoViewer enables fast and efficient NGS assembly browsing, analysis and read mapping. It is highly customized, making it suitable for a wide range of NGS related tasks. Due to its relatively simple architecture, it is easy to add specialised visualization functionalities, facilitating further customised data analysis. The software's source code is freely available; it is open for project and task-specific modifications

Guidelines for optimized gene knockout using CRISPR/Cas9.

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.SCOPUS: re.jinfo:eu-repo/semantics/publishe