The Experts below are selected from a list of 1552593 Experts worldwide ranked by ideXlab platform
Peter J Park - One of the best experts on this subject based on the ideXlab platform.
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ChIP-seq: Advantages and challenges of a maturing technology
Nature Reviews Genetics, 2009Co-Authors: Peter J ParkAbstract:Chromatin immunoprecipitation followed by sequencing (ChIP-seq) is a technique for genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes. Owing to the tremendous progress in next-generation sequencing technology, ChIP-seq offers higher resolution, less noise and greater coverage than its array-based predecessor ChIP-chip. With the decreasing cost of sequencing, ChIP-seq has become an indispensable tool for studying gene regulation and epigenetic mechanisms. In this Review, I describe the benefits and challenges in harnessing this technique with an emphasis on issues related to experimental design and data analysis. ChIP-seq experiments generate large quantities of data, and effective computational analysis will be crucial for uncovering biological mechanisms.
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design and analysis of chip seq experiments for dna binding proteins
Nature Biotechnology, 2008Co-Authors: Peter J Park, Peter V Kharchenko, Michael Y. TolstorukovAbstract:Recent progress in massively parallel sequencing platforms has enabled genome-wide characterization of DNA-associated proteins using the combination of chromatin immunoprecipitation and sequencing (ChIP-seq). Although a variety of methods exist for analysis of the established alternative ChIP microarray (ChIP-chip), few approaches have been described for processing ChIP-seq data. To fill this gap, we propose an analysis pipeline specifically designed to detect protein-binding positions with high accuracy. Using previously reported data sets for three transcription factors, we illustrate methods for improving tag alignment and correcting for background signals. We compare the sensitivity and spatial precision of three peak detection algorithms with published methods, demonstrating gains in spatial precision when an asymmetric distribution of tags on positive and negative strands is considered. We also analyze the relationship between the depth of sequencing and characteristics of the detected binding positions, and provide a method for estimating the sequencing depth necessary for a desired coverage of protein binding sites.
Sandra Cortijo - One of the best experts on this subject based on the ideXlab platform.
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ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy
Tree Genetics and Genomes, 2020Co-Authors: Noemie Vimont, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Fu Xiang Quah, David Guillaume Schöpfer, Sandra CortijoAbstract:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.
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chromatin immunoprecipitation sequencing and rna sequencing for complex and low abundance tree buds
bioRxiv, 2018Co-Authors: Noemie Vimont, David Schoepfer, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Sandra CortijoAbstract:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique combined with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica, Malus x domestica Borkh.) that has also been successfully tested in Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. In this protocol, samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material, differentiating it from previously published protocols. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.
Duncan T Odom - One of the best experts on this subject based on the ideXlab platform.
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mitochondrial heteroplasmy in vertebrates using chip sequencing data
Genome Biology, 2016Co-Authors: Thomas Rensch, Diego Villar, Julie E Horvath, Duncan T Odom, Paul FlicekAbstract:Mitochondrial heteroplasmy, the presence of more than one mitochondrial DNA (mtDNA) variant in a cell or individual, is not as uncommon as previously thought. It is mostly due to the high mutation rate of the mtDNA and limited repair mechanisms present in the mitochondrion. Motivated by mitochondrial diseases, much focus has been placed into studying this phenomenon in human samples and in medical contexts. To place these results in an evolutionary context and to explore general principles of heteroplasmy, we describe an integrated cross-species evaluation of heteroplasmy in mammals that exploits previously reported NGS data. Focusing on ChIP-seq experiments, we developed a novel approach to detect heteroplasmy from the concomitant mitochondrial DNA fraction sequenced in these experiments. We first demonstrate that the sequencing coverage of mtDNA in ChIP-seq experiments is sufficient for heteroplasmy detection. We then describe a novel detection method for accurate detection of heteroplasmies, which also accounts for the error rate of NGS technology. Applying this method to 79 individuals from 16 species resulted in 107 heteroplasmic positions present in a total of 45 individuals. Further analysis revealed that the majority of detected heteroplasmies occur in intergenic regions. In addition to documenting the prevalence of mtDNA in ChIP-seq data, the results of our mitochondrial heteroplasmy detection method suggest that mitochondrial heteroplasmies identified across vertebrates share similar characteristics as found for human heteroplasmies. Although largely consistent with previous studies in individual vertebrates, our integrated cross-species analysis provides valuable insights into the evolutionary dynamics of mitochondrial heteroplasmy.
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chip seq using high throughput sequencing to discover protein dna interactions
Methods, 2009Co-Authors: Dominic Schmidt, Gordon D Brown, Michael D Wilson, Christiana Spyrou, James Hadfield, Duncan T OdomAbstract:Abstract Chromatin immunoprecipitation (ChIP) allows specific protein–DNA interactions to be isolated. Combining ChIP with high-throughput sequencing reveals the DNA sequence involved in these interactions. Here, we describe how to perform ChIP-seq starting with whole tissues or cell lines, and ending with millions of short sequencing tags that can be aligned to the reference genome of the species under investigation. We also outline additional procedures to recover ChIP-chip libraries for ChIP-seq and discuss contemporary issues in data analysis.
Noemie Vimont - One of the best experts on this subject based on the ideXlab platform.
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ChIP-seq and RNA-seq for complex and low-abundance tree buds reveal chromatin and expression co-dynamics during sweet cherry bud dormancy
Tree Genetics and Genomes, 2020Co-Authors: Noemie Vimont, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Fu Xiang Quah, David Guillaume Schöpfer, Sandra CortijoAbstract:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique in combination with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica L Batch, Malus x domestica Borkh.) that has also been successfully tested on Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. Furthermore, one of the advantages of this protocol is that samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material. Focusing on dormant buds in sweet cherry, we explored the link between expression level and H3K4me3 enrichment for all genes, including a strong correlation between H3K4me3 enrichment at the DORMANCY-ASSOCIATED MADS-box 5 (PavDAM5) loci and its expression pattern. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.
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chromatin immunoprecipitation sequencing and rna sequencing for complex and low abundance tree buds
bioRxiv, 2018Co-Authors: Noemie Vimont, David Schoepfer, Francois Roudier, Elisabeth Dirlewanger, Philip A Wigge, Benedicte Wenden, Sandra CortijoAbstract:Chromatin immunoprecipitation-sequencing (ChIP-seq) is a robust technique to study interactions between proteins, such as histones or transcription factors, and DNA. This technique combined with RNA-sequencing (RNA-seq) is a powerful tool to better understand biological processes in eukaryotes. We developed a combined ChIP-seq and RNA-seq protocol for tree buds (Prunus avium L., Prunus persica, Malus x domestica Borkh.) that has also been successfully tested in Arabidopsis thaliana and Saccharomyces cerevisiae. Tree buds contain phenolic compounds that negatively interfere with ChIP and RNA extraction. In addition to solving this problem, our protocol is optimised to work on small amounts of material. In this protocol, samples for ChIP-seq are cross-linked after flash freezing, making it possible to work on trees growing in the field and to perform ChIP-seq and RNA-seq on the same starting material, differentiating it from previously published protocols. This protocol will allow analysis of chromatin and transcriptomic dynamics in tree buds, notably during its development and response to the environment.
B. Jeroen Klevering - One of the best experts on this subject based on the ideXlab platform.
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The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice
Molecular Vision, 2015Co-Authors: Ramon A C Van Huet, Magda A. Meester-smoor, L. Ingeborgh Van Den Born, Laurence Pierrache, Lies H Hoefsloot, Rob W J Collin, Ilse J De Wijs, Caroline C W Klaver, Carel B. Hoyng, B. Jeroen KleveringAbstract:PURPOSE: To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). METHODS: We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon-intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results : The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). CONCLUSIONS: The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis.