Evaluation of experimental design and computational parameter choices affecting analyses of ChIP-seq and RNA-seq data in undomesticated poplar trees.

BackgroundOne of the great advantages of next generation sequencing is the ability to generate large genomic datasets for virtually all species, including non-model organisms. It should be possible, in turn, to apply advanced computational approaches to these datasets to develop models of biological processes. In a practical sense, working with non-model organisms presents unique challenges. In this paper we discuss some of these challenges for ChIP-seq and RNA-seq experiments using the undomesticated tree species of the genus Populus.ResultsWe describe specific challenges associated with experimental design in Populus, including selection of optimal genotypes for different technical approaches and development of antibodies against Populus transcription factors. Execution of the experimental design included the generation and analysis of Chromatin immunoprecipitation-sequencing (ChIP-seq) data for RNA polymerase II and transcription factors involved in wood formation. We discuss criteria for analyzing the resulting datasets, determination of appropriate control sequencing libraries, evaluation of sequencing coverage needs, and optimization of parameters. We also describe the evaluation of ChIP-seq data from Populus, and discuss the comparison between ChIP-seq and RNA-seq data and biological interpretations of these comparisons.ConclusionsThese and other "lessons learned" highlight the challenges but also the potential insights to be gained from extending next generation sequencing-supported network analyses to undomesticated non-model species

Statistical Mutation Calling from Sequenced Overlapping DNA Pools in TILLING Experiments

<p>Abstract</p> <p>Background</p> <p>TILLING (Targeting induced local lesions IN genomes) is an efficient reverse genetics approach for detecting induced mutations in pools of individuals. Combined with the high-throughput of next-generation sequencing technologies, and the resolving power of overlapping pool design, TILLING provides an efficient and economical platform for functional genomics across thousands of organisms.</p> <p>Results</p> <p>We propose a probabilistic method for calling TILLING-induced mutations, and their carriers, from high throughput sequencing data of overlapping population pools, where each individual occurs in two pools. We assign a probability score to each sequence position by applying Bayes' Theorem to a simplified binomial model of sequencing error and expected mutations, taking into account the coverage level. We test the performance of our method on variable quality, high-throughput sequences from wheat and rice mutagenized populations.</p> <p>Conclusions</p> <p>We show that our method effectively discovers mutations in large populations with sensitivity of 92.5% and specificity of 99.8%. It also outperforms existing SNP detection methods in detecting real mutations, especially at higher levels of coverage variability across sequenced pools, and in lower quality short reads sequence data. The implementation of our method is available from: <url>http://www.cs.ucdavis.edu/filkov/CAMBa/</url>.</p

High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants

Genomic Analysis of Parent-of-Origin Allelic Expression in Arabidopsis thaliana Seeds

Differential expression of maternally and paternally inherited alleles of a gene is referred to as gene imprinting, a form of epigenetic gene regulation common to flowering plants and mammals. In plants, imprinting primarily occurs in the endosperm, a seed tissue that supports the embryo during its growth and development. Previously, we demonstrated that widespread DNA demethylation at remnants of transposable elements accompanies endosperm development and that a subset of these methylation changes are associated with gene imprinting. Here we assay imprinted gene expression genome-wide by performing high-throughput sequencing of RNA derived from seeds of reciprocal intraspecific crosses. We identify more than 200 loci that exhibit parent-of-origin effects on gene expression in the endosperm, including a large number of transcription factors, hormone biosynthesis and response genes, and genes that encode regulators of epigenetic information, such as methylcytosine binding proteins, histone methyltransferases, and chromatin remodelers. The majority of these genes are partially, rather than completely, imprinted, suggesting that gene dosage regulation is an important aspect of imprinted gene expression

Evaluation of experimental design and computational parameter choices affecting analyses of ChIP-seq and RNA-seq data in undomesticated poplar trees.

BackgroundOne of the great advantages of next generation sequencing is the ability to generate large genomic datasets for virtually all species, including non-model organisms. It should be possible, in turn, to apply advanced computational approaches to these datasets to develop models of biological processes. In a practical sense, working with non-model organisms presents unique challenges. In this paper we discuss some of these challenges for ChIP-seq and RNA-seq experiments using the undomesticated tree species of the genus Populus.ResultsWe describe specific challenges associated with experimental design in Populus, including selection of optimal genotypes for different technical approaches and development of antibodies against Populus transcription factors. Execution of the experimental design included the generation and analysis of Chromatin immunoprecipitation-sequencing (ChIP-seq) data for RNA polymerase II and transcription factors involved in wood formation. We discuss criteria for analyzing the resulting datasets, determination of appropriate control sequencing libraries, evaluation of sequencing coverage needs, and optimization of parameters. We also describe the evaluation of ChIP-seq data from Populus, and discuss the comparison between ChIP-seq and RNA-seq data and biological interpretations of these comparisons.ConclusionsThese and other "lessons learned" highlight the challenges but also the potential insights to be gained from extending next generation sequencing-supported network analyses to undomesticated non-model species

High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core "DSB" response specific to clastogenic treatments.

Plants exhibit a robust transcriptional response to gamma radiation which includes the induction of transcripts required for homologous recombination and the suppression of transcripts that promote cell cycle progression. Various DNA damaging agents induce different spectra of DNA damage as well as "collateral" damage to other cellular components and therefore are not expected to provoke identical responses by the cell. Here we study the effects of two different types of ionizing radiation (IR) treatment, HZE (1 GeV Fe(26+) high mass, high charge, and high energy relativistic particles) and gamma photons, on the transcriptome of Arabidopsis thaliana seedlings. Both types of IR induce small clusters of radicals that can result in the formation of double strand breaks (DSBs), but HZE also produces linear arrays of extremely clustered damage. We performed these experiments across a range of time points (1.5-24 h after irradiation) in both wild-type plants and in mutants defective in the DSB-sensing protein kinase ATM. The two types of IR exhibit a shared double strand break-repair-related damage response, although they differ slightly in the timing, degree, and ATM-dependence of the response. The ATM-dependent, DNA metabolism-related transcripts of the "DSB response" were also induced by other DNA damaging agents, but were not induced by conventional stresses. Both Gamma and HZE irradiation induced, at 24 h post-irradiation, ATM-dependent transcripts associated with a variety of conventional stresses; these were overrepresented for pathogen response, rather than DNA metabolism. In contrast, only HZE-irradiated plants, at 1.5 h after irradiation, exhibited an additional and very extensive transcriptional response, shared with plants experiencing "extended night." This response was not apparent in gamma-irradiated plants

Corrigendum: High atomic weight, high-energy radiation (HZE) induces transcriptional responses shared with conventional stresses in addition to a core “DSB” response specific to clastogenic treatments

[This corrects the article on p. 364 in vol. 5, PMID: 25136344.]