A cell epigenotype specific model for the correction of brain cellular heterogeneity bias and its application to age, brain region and major depression

Brain cellular heterogeneity may bias cell type specific DNA methylation patterns, influencing findings in psychiatric epigenetic studies. We performed fluorescence activated cell sorting (FACS) of neuronal nuclei and Illumina HM450 DNA methylation profiling in post mortem frontal cortex of 29 major depression and 29 matched controls. We identify genomic features and ontologies enriched for cell type specific epigenetic variation. Using the top cell epigenotype specific (CETS) marks, we generated a publically available R package, “CETS,” capable of quantifying neuronal proportions and generating in silico neuronal profiles from DNA methylation data. We demonstrate a significant overlap in major depression DNA methylation associations between FACS separated and CETS model generated neuronal profiles relative to bulk profiles. CETS derived neuronal proportions correlated significantly with age in the frontal cortex and cerebellum and accounted for epigenetic variation between brain regions. CETS based control of cellular heterogeneity will enable more robust hypothesis testing in the brain

Identification of direction in gene networks from expression and methylation

Background: Reverse-engineering gene regulatory networks from expression data is difficult, especially without temporal measurements or interventional experiments. In particular, the causal direction of an edge is generally not statistically identifiable, i.e., cannot be inferred as a statistical parameter, even from an unlimited amount of non-time series observational mRNA expression data. Some additional evidence is required and high-throughput methylation data can viewed as a natural multifactorial gene perturbation experiment. Results: We introduce IDEM (Identifying Direction from Expression and Methylation), a method for identifying the causal direction of edges by combining DNA methylation and mRNA transcription data. We describe the circumstances under which edge directions become identifiable and experiments with both real and synthetic data demonstrate that the accuracy of IDEM for inferring both edge placement and edge direction in gene regulatory networks is significantly improved relative to other methods. Conclusion: Reverse-engineering directed gene regulatory networks from static observational data becomes feasible by exploiting the context provided by high-throughput DNA methylation data. An implementation of the algorithm described is available at http://code.google.com/p/idem/

diffloop: a computational framework for identifying and analyzing differential DNA loops from sequencing data

Abstract Summary The 3D architecture of DNA within the nucleus is a key determinant of interactions between genes, regulatory elements, and transcriptional machinery. As a result, differences in DNA looping structure are associated with variation in gene expression and cell state. To systematically assess changes in DNA looping architecture between samples, we introduce diffloop, an R/Bioconductor package that provides a suite of functions for the quality control, statistical testing, annotation, and visualization of DNA loops. We demonstrate this functionality by detecting differences between ENCODE ChIA-PET samples and relate looping to variability in epigenetic state. Availability and implementation Diffloop is implemented as an R/Bioconductor package available at https://bioconductor.org/packages/release/bioc/html/diffloop.html Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online

An improved empirical bayes approach to estimating differential gene expression in microarray time-course data: BETR (Bayesian Estimation of Temporal Regulation)

<p>Abstract</p> <p>Background</p> <p>Microarray gene expression time-course experiments provide the opportunity to observe the evolution of transcriptional programs that cells use to respond to internal and external stimuli. Most commonly used methods for identifying differentially expressed genes treat each time point as independent and ignore important correlations, including those within samples and between sampling times. Therefore they do not make full use of the information intrinsic to the data, leading to a loss of power.</p> <p>Results</p> <p>We present a flexible random-effects model that takes such correlations into account, improving our ability to detect genes that have sustained differential expression over more than one time point. By modeling the joint distribution of the samples that have been profiled across all time points, we gain sensitivity compared to a marginal analysis that examines each time point in isolation. We assign each gene a probability of differential expression using an empirical Bayes approach that reduces the effective number of parameters to be estimated.</p> <p>Conclusions</p> <p>Based on results from theory, simulated data, and application to the genomic data presented here, we show that BETR has increased power to detect subtle differential expression in time-series data. The open-source R package <it>betr </it>is available through Bioconductor. BETR has also been incorporated in the freely-available, open-source MeV software tool available from <url>http://www.tm4.org/mev.html</url>.</p

Detection, quantification and genotyping of Herpes Simplex Virus in cervicovaginal secretions by real-time PCR: a cross sectional survey

BACKGROUND: Herpes Simplex Virus (HSV) Genital Ulcer Disease (GUD) is an important public health problem, whose interaction with HIV results in mutually enhancing epidemics. Conventional methods for detecting HSV tend to be slow and insensitive. We designed a rapid PCR-based assay to quantify and type HSV in cervicovaginal lavage (CVL) fluid of subjects attending a Genito-Urinary Medicine (GUM) clinic. Vaginal swabs, CVL fluid and venous blood were collected. Quantitative detection of HSV was conducted using real time PCR with HSV specific primers and SYBR Green I. Fluorogenic TaqMan Minor Groove Binder (MGB) probes designed around a single base mismatch in the HSV DNA polymerase I gene were used to type HSV in a separate reaction. The Kalon test was used to detect anti-HSV-2 IgG antibodies in serum. Testing for HIV, other Sexually Transmitted Infections (STI) and related infections was based on standard clinical and laboratory methods. RESULTS: Seventy consecutive GUM clinic attendees were studied. Twenty-seven subjects (39%) had detectable HSV DNA in CVL fluid; HSV-2 alone was detected in 19 (70%) subjects, HSV-1 alone was detected in 4 (15%) subjects and both HSV types were detected in 4 (15%) subjects. Eleven out of 27 subjects (41%) with anti-HSV-2 IgG had detectable HSV-2 DNA in CVL fluid. Seven subjects (10%) were HIV-positive. Three of seven (43%) HIV-infected subjects and two of five subjects with GUD (40%) were secreting HSV-2. None of the subjects in whom HSV-1 was detected had GUD. CONCLUSION: Quantitative real-time PCR and Taqman MGB probes specific for HSV-1 or -2 were used to develop an assay for quantification and typing of HSV. The majority of subjects in which HSV was detected had low levels of CVL fluid HSV, with no detectable HSV-2 antibodies and were asymptomatic

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing

Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI Nucleases (RFNs) that recognize extended sequences and can edit endogenous genes with high efficiencies in human cells. The cleavage activity of an RFN depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation and therefore show improved specificities relative to wild-type Cas9 monomers. Importantly, direct comparisons show that RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5′ end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing

Altering, Improving, And Defining The Specificities Of Crispr-Cas Nucleases

CRISPR-Cas9 nucleases have been widely adopted for genome editing applications to knockout genes or to introduce desired changes. While these nucleases have shown immense promise, two notable limitations of the wild-type form of the broadly used Streptococcus pyogenes Cas9 (SpCas9) are the restriction of targeting range to sites that contain an NGG protospacer adjacent motif (PAM), and the undesirable ability of the enzyme to cleave off-target sites that resemble the on-target site. Scarcity of PAM motifs can limit implementations that require precise targeting, whereas off-target effects can confound research applications and are important considerations for therapeutics. To improve the targeting range of SpCas9 and an orthogonal Cas9 from Staphylococcus aureus (called SaCas9), we optimized a heterologous genetic selection system that enabled us to perform directed evolution of PAM specificity. With SpCas9, we evolved two separate variants that can target NGA and NGCG PAMs1, and with SaCas9 relaxed the PAM from NNGRRT to NNNRRT2, increasing the targetability of these enzyme 2- to 4-fold. The genome-wide specificity profiles of SpCas9 and SaCas9 variants, determine by GUIDE-seq3, indicate that they are at least as, if not more, specific than the wild-type enzyme1,2. Together, these results demonstrate that the inherent PAM specificity of multiple different Cas9 orthologues can be purposefully modified to improve the accuracy of targeting. Existing strategies for improving the genome-wide specificity of SpCas9 have thus far proven to be incompletely effective and/or have other limitations that constrain their use. To address the off-target potential of SpCas9, we engineered a high-fidelity variant of SpCas9 (called SpCas9-HF1), that contains alterations designed to reduce non-specific contacts to the target strand DNA backbone. In comparison to wild-type SpCas9, SpCas9-HF1 rendered all or nearly all off-target events imperceptible by GUIDE-seq and targeted deep-sequencing methods with standard non-repetitive target sites in human cells4. Even for atypical, repetitive target sites, the vast majority of off-targets induced by SpCas9-HF1 and optimized derivatives were not detected4. With its exceptional precision, SpCas9-HF1 provides an important and easily employed alternative to wild-type SpCas9 that can eliminate off-target effects when using CRISPR-Cas9 for research and therapeutic applications. Finally, on-target activity and genome-wide specificity are two important properties of engineered nucleases that should be characterized prior to adoption of such technologies for research or therapeutic applications. CRISPR-Cas Cpf1 nucleases have recently been described as an alternative genome-editing platform5, yet their activities and genome-wide specificities remain largely undefined. Based on assessment of on-target activity across more than 40 target sites, we demonstrate that two Cpf1 orthologues function robustly in human cells with efficiencies comparable to those of the widely used Streptococcus pyogenes Cas9. We also demonstrate that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 are insensitive to single base mismatches, but that many of the other bases within the crRNA targeting region are highly sensitive to single or double substitutions6. Consistent with these results, GUIDE-seq performed in multiple cell types and targeted deep sequencing analyses of two Cpf1 nucleases revealed no detectable off-target cleavage for over half of 20 different crRNAs we examined. Our results suggest that the two Cpf1 nucleases we characterized generally possess robust on-target activity and high specificities in human cells, findings that should encourage broader use of these genome editing enzymes. 1. Kleinstiver, BP, et al. (2015) Nature, 523(7561):481-5 2. Kleinstiver, BP, et al. (2015) Nature Biotechnology, 33(12):1293-98 3. Tsai, SQ et al. (2015) Nature Biotechnology, 33(2):187-97 4. Kleinstiver, BP and Pattanayak, V, et al. (2016), Nature, 529(7587):490-5 5. Zetsche, B, et al. (2015) Cell, 163(3):759-71 6. Kleinstiver, BP and Tsai, SQ, et al. (2016), Nature Biotechnology, 34(8):869-7

Comprehensive methylome map of lineage commitment from haematopoietic progenitors.

Epigenetic modifications must underlie lineage-specific differentiation as terminally differentiated cells express tissue-specific genes, but their DNA sequence is unchanged. Haematopoiesis provides a well-defined model to study epigenetic modifications during cell-fate decisions, as multipotent progenitors (MPPs) differentiate into progressively restricted myeloid or lymphoid progenitors. Although DNA methylation is critical for myeloid versus lymphoid differentiation, as demonstrated by the myeloerythroid bias in Dnmt1 hypomorphs, a comprehensive DNA methylation map of haematopoietic progenitors, or of any multipotent/oligopotent lineage, does not exist. Here we examined 4.6 million CpG sites throughout the genome for MPPs, common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte/macrophage progenitors (GMPs), and thymocyte progenitors (DN1, DN2, DN3). Marked epigenetic plasticity accompanied both lymphoid and myeloid restriction. Myeloid commitment involved less global DNA methylation than lymphoid commitment, supported functionally by myeloid skewing of progenitors following treatment with a DNA methyltransferase inhibitor. Differential DNA methylation correlated with gene expression more strongly at CpG island shores than CpG islands. Many examples of genes and pathways not previously known to be involved in choice between lymphoid/myeloid differentiation have been identified, such as Arl4c and Jdp2. Several transcription factors, including Meis1, were methylated and silenced during differentiation, indicating a role in maintaining an undifferentiated state. Additionally, epigenetic modification of modifiers of the epigenome seems to be important in haematopoietic differentiation. Our results directly demonstrate that modulation of DNA methylation occurs during lineage-specific differentiation and defines a comprehensive map of the methylation and transcriptional changes that accompany myeloid versus lymphoid fate decisions

Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background DNA methylation has been linked to genome regulation and dysregulation in health and disease respectively, and methods for characterizing genomic DNA methylation patterns are rapidly emerging. We have developed/refined methods for enrichment of methylated genomic fragments using the methyl-binding domain of the human MBD2 protein (MBD2-MBD) followed by analysis with high-density tiling microarrays. This MBD-chip approach was used to characterize DNA methylation patterns across all non-repetitive sequences of human chromosomes 21 and 22 at high-resolution in normal and malignant prostate cells. Results Examining this data using computational methods that were designed specifically for DNA methylation tiling array data revealed widespread methylation of both gene promoter and non-promoter regions in cancer and normal cells. In addition to identifying several novel cancer hypermethylated 5' gene upstream regions that mediated epigenetic gene silencing, we also found several hypermethylated 3' gene downstream, intragenic and intergenic regions. The hypermethylated intragenic regions were highly enriched for overlap with intron-exon boundaries, suggesting a possible role in regulation of alternative transcriptional start sites, exon usage and/or splicing. The hypermethylated intergenic regions showed significant enrichment for conservation across vertebrate species. A sampling of these newly identified promoter (ADAMTS1 and SCARF2 genes) and non-promoter (downstream or within DSCR9, C21orf57 and HLCS genes) hypermethylated regions were effective in distinguishing malignant from normal prostate tissues and/or cell lines. Conclusions Comparison of chromosome-wide DNA methylation patterns in normal and malignant prostate cells revealed significant methylation of gene-proximal and conserved intergenic sequences. Such analyses can be easily extended for genome-wide methylation analysis in health and disease.Published versio

Diarrhea incidence in low- and middle-income countries in 1990 and 2010: a systematic review

<p>Abstract</p> <p>Background</p> <p>Diarrhea is recognized as a leading cause of morbidity and mortality among children under 5 years of age in low- and middle-income countries yet updated estimates of diarrhea incidence by age for these countries are greatly needed. We conducted a systematic literature review to identify cohort studies that sought to quantify diarrhea incidence among any age group of children 0-59 mo of age.</p> <p>Methods</p> <p>We used the Expectation-Maximization algorithm as a part of a two-stage regression model to handle diverse age data and overall incidence rate variation by study to generate country specific incidence rates for low- and middle-income countries for 1990 and 2010. We then calculated regional incidence rates and uncertainty ranges using the bootstrap method, and estimated the total number of episodes for children 0-59 mo of age in 1990 and 2010.</p> <p>Results</p> <p>We estimate that incidence has declined from 3.4 episodes/child year in 1990 to 2.9 episodes/child year in 2010. As was the case previously, incidence rates are highest among infants 6-11 mo of age; 4.5 episodes/child year in 2010. Among these 139 countries there were nearly 1.9 billion episodes of childhood diarrhea in 1990 and nearly 1.7 billion episodes in 2010.</p> <p>Conclusions</p> <p>Although our results indicate that diarrhea incidence rates may be declining slightly, the total burden on the health of each child due to multiple episodes per year is tremendous and additional funds are needed to improve both prevention and treatment practices in low- and middle-income countries.</p