ATAC-seq Experiments Research Articles

Abstract 17503: ETV2 Functions as a Pioneer Factor and Regulates the Endothelial Lineage

Background: Genetic mutations perturb the multipotent progenitors, which results in congenital cardiovascular disease. Therefore, it is essential to decipher the pioneer factors and the regulatory pathways that govern the specification and differentiation of mesodermal progenitors and use this information to develop targeted therapies to promote cardiovascular regeneration. Etv2 as an essential transcription factor for the development of cardiac, endothelial and hematopoietic lineages. In the present study, we used ES/EB differentiation and MEF reprogramming systems, to define Etv2 as a novel pioneer factor that relaxes the closed chromatin and drives endothelial development. Results: Using the iHA-Etv2 ES cell line, we engineered a mouse that inducibly overexpresses ETV2. The bulk RNA-seq, single cell RNA-seq data and ATAC-seq experiments showed that inducing Etv2 in MEFs and ES/EBs activated the downstream endothelial marker genes and promoted the development of endothelial lineages, supporting the notion that Etv2 functioned as a master regulator to drive the endothelial lineage development in different cellular contexts. We found that similar to other known pioneer factors, Etv2 was intrinsically able to target and bind the nucleosomes, and this capability appeared to be independent of the cellular context. To further define the mechanism, we performed Etv2, Brg1 and H3K27ac ChIP-seq analyses during MEF reprogramming and ES/EB differentiation. We found that Brg1 maintains and stabilizes the binding of Etv2 on the nucleosome, and Etv2 requires Brg1 to activate downstream genes during reprograming. Conclusion: In these studies, we defined Etv2 as a novel pioneer factor that relaxed the closed chromatin and promoted the endothelial lineage in both ES/EB differentiation and MEF reprogramming. The definition of these mechanisms will enhance our understanding of cardiovascular development and regeneration and serve as a platform for therapeutic applications for patients with congenital or aging related cardiovascular diseases.

Decoding myofibroblast origins in human kidney fibrosis.

Kidney fibrosis is the hallmark of chronic kidney disease progression, however, currently no antifibrotic therapies exist. This is largely because the origin, functional heterogeneity and regulation of scar-forming cells during human kidney fibrosis remains poorly understood. Here, using single cell RNA-seq, we profiled the transcriptomes of proximal tubule and non-proximal tubule cells in healthy and fibrotic human kidneys to map the entire human kidney in an unbiased approach. This enabled mapping of all matrix-producing cells at high resolution, revealing distinct subpopulations of pericytes and fibroblasts as the major cellular sources of scar forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single cell RNA-seq and ATAC-seq experiments in mice, and spatial transcriptomics in human kidney fibrosis to functionally interrogate these findings, shedding new light on the origin, heterogeneity and differentiation of human kidney myofibroblasts and their fibroblast and pericyte precursors at unprecedented resolution. Finally, we used this strategy to facilitate target discovery, identifying Nkd2 as a myofibroblast-specific target in human kidney fibrosis.

2018 – EOMESODERMIN GOVERNS THE HEMOGENIC COMPETENCE OF MURINE YOLK-SAC MESODERMAL PROGENITORS

The gene regulatory networks that coordinate hematopoietic commitment in the developing murine yolk-sac (YS) remain ill-defined. Here we report that the T-box transcription factor Eomesodermin (Eomes) is transiently expressed in mesodermal progenitors that generate virtually all YS hematopoietic and endothelial cells. Using an embryonic stem cell (ESC) differentiation system, we find that Eomes activity is essential for the production of primitive erythrocytes and definitive hematopoietic progenitors but dispensable for the development of endothelial cells. Bulk RNA-seq and single-cell-RNA-seq experiments demonstrate that in the absence of Eomes function Flk-1+ hematovascular mesoderm is specified but upon further differentiation it is diverted towards an endothelial rather than hematopoietic fate. Utilizing ESC reporter lines, we show that Eomes is expressed prior to both SCL and Runx1 during mesoderm patterning. Interestingly, Eomes activity is dispensable for the expression of SCL but is essential for the normal development of Runx1+ hemogenic endothelium (HE). ATAC-Seq experiments reveal that Eomes governs the accessibility of numerous hematopoietic enhancers that SCL normally utilizes to specify the hematopoietic fate. Finally, ChIP-seq experiments suggest that Eomes coordinates the development of hemogenic competent mesoderm in the context of Activin/Nodal and Tead-Yap signalling at mesodermal stages of development. Collectively, these experiments demonstrate that Eomes sits at the top of the transcriptional hierarchy, functioning upstream of Runx1 expression and SCL functional activity, to promote hemogenic competence of the YS mesodermal lineage. These results suggest that hemogenic competence is endowed earlier during murine embryogenesis than previously appreciated and have widespread implications for the generation of HE from pluripotent stem cell sources.

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method.

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension withintact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

303 Allele specific accessibility analysis to decipher molecular mechanism of psoriasis-associated loci

Genome-wide association studies for psoriasis have identified >65 susceptibility regions, and our previous work has revealed these genetic signals overlap significantly with active enhancers in T-cells. To better understand the molecular mechanisms underlying the regulatory roles of these genetic signals, we conducted ATAC-seq to profile the chromatin accessibility patterns of resting and 24h CD3/CD28-activated T-cell subsets from >100 individuals. By tallying ATAC-seq read counts as a function of allelic variation, we assessed allele specific accessibility (ASA) for genetic variants mapping within +/- 100 kb of the 95% credible set of causal variants for the psoriasis-associated loci. By considering the directionality of the accessibility of the allele and information from multiple samples, we identified >700 sites with heterozygous genotypes and mapping to accessible chromatin in the ATAC-seq experiments. Genome-wide, we found stronger ASA in open chromatin regions depending on their proximity to psoriasis-associated signals. We further identified at least 9 psoriasis-associated loci encompassing markers exhibiting significant (FDR<=10%) ASA, including IL28RAand ZNF365, both of which manifested ASA in activated but not resting T-cells. By jointly leveraging sequence variation and chromatin accessibility information, our results provide novel insights to the molecular mechanisms of psoriasis-associated genetic variation. Ongoing sample size expansion and GWAS genotyping will add to the catalog of psoriasis-associated variants manifesting context-dependent ASA in T-cells, generating new hypotheses for functional and mechanistic studies.

H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles.

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

Testing Proximity of Genomic Regions to Transcription Start Sites and Enhancers Complements Gene Set Enrichment Testing.

Large sets of genomic regions are generated by the initial analysis of various genome-wide sequencing data, such as ChIP-seq and ATAC-seq experiments. Gene set enrichment (GSE) methods are commonly employed to determine the pathways associated with them. Given the pathways and other gene sets (e.g., GO terms) of significance, it is of great interest to know the extent to which each is driven by binding near transcription start sites (TSS) or near enhancers. Currently, no tool performs such an analysis. Here, we present a method that addresses this question to complement GSE methods for genomic regions. Specifically, the new method tests whether the genomic regions in a gene set are significantly closer to a TSS (or to an enhancer) than expected by chance given the total list of genomic regions, using a non-parametric test. Combining the results from a GSE test with our novel method provides additional information regarding the mode of regulation of each pathway, and additional evidence that the pathway is truly enriched. We illustrate our new method with a large set of ENCODE ChIP-seq data, using the chipenrich Bioconductor package. The results show that our method is a powerful complementary approach to help researchers interpret large sets of genomic regions.

Abstract IA20: Chromatin state and scRNA-seq analysis defines a common differentiation trajectory towards T-cell exhaustion

Abstract We will describe a unified chromatin state and single cell expression analysis underlying T-cell differentiation to “exhaustion.” Numerous studies in chronic viral infection and tumor models have shown that most T cells progress to a terminally dysfunctional (“exhausted”) state from which they cannot be rescued by current immunotherapies. We performed a systematic analysis of over 280 ATAC-seq and RNA-seq experiments from eight published studies of CD8 T cell dysfunction, using a statistical batch correction to define a common differentiation trajectory to terminal exhaustion in all settings of chronic antigen exposure and to identify transcription factors involved in this progression. We further performed scRNA-seq analysis of CD8 T cells at multiple time points during acute and chronic viral infection and in adoptive cell transfer studies to elucidate this trajectory at single-cell resolution. Citation Format: Christina Leslie. Chromatin state and scRNA-seq analysis defines a common differentiation trajectory towards T-cell exhaustion [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr IA20.

CASZ1 induces skeletal muscle and rhabdomyosarcoma differentiation through a feed-forward loop with MYOD and MYOG

Embryonal rhabdomyosarcoma (ERMS) is a childhood cancer that expresses myogenic master regulatory factor MYOD but fails to differentiate. Here, we show that the zinc finger transcription factor CASZ1 up-regulates MYOD signature genes and induces skeletal muscle differentiation in normal myoblasts and ERMS. The oncogenic activation of the RAS-MEK pathway suppresses CASZ1 expression in ERMS. ChIP-seq, ATAC-seq and RNA-seq experiments reveal that CASZ1 directly up-regulates skeletal muscle genes and represses non-muscle genes through affecting regional epigenetic modifications, chromatin accessibility and super-enhancer establishment. Next generation sequencing of primary RMS tumors identified a single nucleotide variant in the CASZ1 coding region that potentially contributes to ERMS tumorigenesis. Taken together, loss of CASZ1 activity, due to RAS-MEK signaling or genetic alteration, impairs ERMS differentiation, contributing to RMS tumorigenesis.

Fluent genomics with plyrangesand tximeta.

We construct a simple workflow for fluent genomics data analysis using the R/Bioconductor ecosystem. This involves three core steps: importthe data into an appropriate abstraction, model the data with respect to the biological questions of interest, and integrate the results with respect to their underlying genomic coordinates. Here we show how to implement these steps to integrate published RNA-seq and ATAC-seq experiments on macrophage cell lines. Using tximeta, we importRNA-seq transcript quantifications into an analysis-ready data structure, called the SummarizedExperiment, that contains the ranges of the reference transcripts and metadata on their provenance. Using SummarizedExperiments to represent the ATAC-seq and RNA-seq data, we model differentially accessible (DA) chromatin peaks and differentially expressed (DE) genes with existing Bioconductor packages. Using plyrangeswe then integratethe results to see if there is an enrichment of DA peaks near DE genes by finding overlaps and aggregating over log-fold change thresholds. The combination of these packages and their integration with the Bioconductor ecosystem provide a coherent framework for analysts to iteratively and reproducibly explore their biological data.

Higher Order Organization of the mtDNA: Beyond Mitochondrial Transcription Factor A.

The higher order organization of eukaryotic and prokaryotic genomes is pivotal in the regulation of gene expression. Specifically, chromatin accessibility in eukaryotes and nucleoid accessibility in bacteria are regulated by a cohort of proteins to alter gene expression in response to diverse physiological conditions. By contrast, prior studies have suggested that the mitochondrial genome (mtDNA) is coated solely by mitochondrial transcription factor A (TFAM), whose increased cellular concentration was proposed to be the major determinant of mtDNA packaging in the mitochondrial nucleoid. Nevertheless, recent analysis of DNase-seq and ATAC-seq experiments from multiple human and mouse samples suggest gradual increase in mtDNA occupancy during the course of embryonic development to generate a conserved footprinting pattern which correlate with sites that have low TFAM occupancy in vivo (ChIP-seq) and tend to adopt G-quadruplex structures. These findings, along with recent identification of mtDNA binding by known modulators of chromatin accessibility such as MOF, suggest that mtDNA higher order organization is generated by cross talk with the nuclear regulatory system, may have a role in mtDNA regulation, and is more complex than once thought.

ATAC-pipe: general analysis of genome-wide chromatin accessibility.

Assay of Transposase-Accessible Chromatin by deep sequencing (ATAC-seq) has been widely used to profile the chromatin accessibility genome-wide. For the absence of an integrated scheme for deep data mining of specific biological issues, here we present ATAC-pipe, an efficient pipeline for general analysis of chromatin accessibility data obtained from ATAC-seq experiments. ATAC-pipe captures information includes not only the quality of original data and genome-wide chromatin accessibility but also signatures of significant differential peaks, transcription factor (TF) occupancy and nucleosome positions around regulatory sites. In addition, ATAC-pipe automatically converts statistic results into intuitive plots at publication quality, such as the read length distribution, heatmaps of sample clustering and cell-type-specific regulatory elements, enriched TF occupancy with motifs footprints and TF-driven regulatory networks. ATAC-pipe provides convenient workflow for researchers to study chromatin accessibility and gene regulation. Availability https://github.com/QuKunLab/ATAC-pipe.

Recurrent Mutations in EGR2 Direct Specific Epigenetic Reconfiguration in Chronic Lymphocytic Leukemia

Epigenetic alterations are universal in cancer and are important in establishing the malignant phenotype. Dissection of the factors that shape the tumor-specific epigenome may reveal insight into key aspects of tumorigenesis and therapeutic resistance. In chronic lymphocytic leukemia (CLL), we have previously found that broad changes in epigenetic patterns co-occur with the evolution of genetic alterations. We have also uncovered that aberrant patterning of DNA methylation in CLL involves excessive activity of a defined group of transcription factors (TFs), including the early growth response (EGR) TF family. Recent work has further revealed that recurrent mutations in EGR2 are associated with exceptionally poor clinical outcomes in CLL. The basis for the adverse association of EGR2 mutations in CLL is unclear.To explore the role of EGR2 mutations in CLL, we initially performed genome-wide DNA methylation analysis using Illumina arrays on CLL patients harboring EGR2 mutations (n=27) compared to EGR wild-type cases (n=265). We found that the three most common recurrent mutations, occurring at amino acid positions E356K, H384N and D411H within the DNA binding domain, are each associated with an exclusive subset of tumor-specific hypomethylated CpG sites. A search for TF sequence motifs at these loci revealed a strong enrichment of novel derivative EGR2 motifs that differ only marginally (usually by a single nucleotide) from the canonical EGR2 recognition sequence. Each recurrent mutation led to specific enrichment of a different derivative EGR2 motif. Furthermore, the canonical (wild-type) recognition sequence was not enriched, suggesting that mutations re-localize binding activity to derivate sequence motifs rather than simply altering binding affinity. Luciferase enhancer, proximity ligation and electrophoretic mobility shift assays confirmed that each EGR2 mutant protein specifically binds and enhances transcriptional activity only when the matched EGR2 derivative recognition motif is present. These results establish that derivative motif sequences may function as novel cryptic enhancers in the presence of the cognate EGR2 mutant TF.We performed multiomics profiling (DNA methylome, ATAC-seq, ChIP-seq and RNA-seq) to examine the nature of the epigenetic reconfiguration and the phenotypic impact of individual EGR2 mutations. Whole genome bisulfite sequencing of E356K- and H384N-mutated CLL samples (n=4 each) was used to reveal the full complement of recurrent differentially methylated regions (DMRs) across the genome, and recapitulated the mutually-exclusive pattern of DMRs between mutations. Overlaying DMRs with data from ChIP-seq and ATAC-seq experiments in the same samples revealed the nature of EGR2 mutation-specific chromatin reconfiguration to be remarkably mutation-specific. For E356K, hypomethylated DMRs are often associated with foci of accessible chromatin, EGR2 binding, and flanked by gains of H3K4me1 and H3K27ac, indicative of the acquisition of active enhancer function. Conversely, H384N mutations generated fewer DMRs and mainly directed the deposition of H3K4me1 only, indicative of gain of poised enhancers at these loci. RNA-sequencing analyses revealed that a subset of epigenetically reconfigured regions was associated with mutation-specific altered gene expression, and differences were virtually always associated with proximal gene activation. E356K and H384N mutations displayed highly differential gene expression patterns, with E356K exhibiting a greater impact on gene expression. Integrated analyses indicated that E356K mutations may specifically involve activated Notch signaling, revealed by the aberrant activation of Notch target genes and the mutual exclusivity of NOTCH1 mutations, further highlighted by enriched co-mutation of NOTCH1 in H384N-mutated CLL.Together these findings provide an exceptional example of the precise role that a singular TF may play in programming the epigenetic landscape. As there are no known TFs that naturally bind derivative EGR2 motifs, these mutant proteins provide insight into aberrant enhancer generation and the phenotypic impact of (re)directed TF binding in a human disease setting. Although these recurrent mutations are presently only known in CLL, these findings provide insight into the mechanisms that may surround other gain-of-function TF activity in various malignancies. DisclosuresKipps:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; Genentech Inc: Consultancy, Research Funding; Pharmacyclics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy; F. Hoffmann-La Roche Ltd: Consultancy, Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees.

Genome-wide chromatin accessibility and transcriptome profiling show minimal epigenome changes and coordinated transcriptional dysregulation of hedgehog signaling in Danforth's short tail mice.

Danforth's short tail (Sd) mice provide an excellent model for investigating the underlying etiology of human caudal birth defects, which affect 1 in 10000 live births. Sd animals exhibit aberrant axial skeleton, urogenital and gastrointestinal development similar to human caudal malformation syndromes including urorectal septum malformation, caudal regression, vertebral-anal-cardiac-tracheo-esophageal fistula-renal-limb (VACTERL) association and persistent cloaca. Previous studies have shown that the Sd mutation results from an endogenous retroviral (ERV) insertion upstream of the Ptf1a gene resulting in its ectopic expression at E9.5. Though the genetic lesion has been determined, the resulting epigenomic and transcriptomic changes driving the phenotype have not been investigated. Here, we performed ATAC-seq experiments on isolated E9.5 tailbud tissue, which revealed minimal changes in chromatin accessibility in Sd/Sd mutant embryos. Interestingly, chromatin changes were localized to a small interval adjacent to the Sd ERV insertion overlapping a known Ptf1a enhancer region, which is conserved in mice and humans. Furthermore, mRNA-seq experiments revealed increased transcription of Ptf1a target genes and, importantly, downregulation of hedgehog pathway genes. Reduced sonic hedgehog (SHH) signaling was confirmed by in situ hybridization and immunofluorescence suggesting that the Sd phenotype results, in part, from downregulated SHH signaling. Taken together, these data demonstrate substantial transcriptome changes in the Sd mouse, and indicate that the effect of the ERV insertion on Ptf1a expression may be mediated by increased chromatin accessibility at a conserved Ptf1a enhancer. We propose that human caudal dysgenesis disorders may result from dysregulation of hedgehog signaling pathways.

Abstract 3353: ZNF414 as a functionally relevant transcription factor in pancreatic and breast cancer cells

Abstract Background: The Zinc Finger Protein 414 (ZNF414) is a member of the krüppel C2H2-type zinc-finger protein family. ZNF414 is a cargo protein for Karyopherin α7 (KPNA7), a nuclear importer expressed during embryogenesis, absent in most adult tissues but re-expressed in cancer cells. KPNA7 is involved in promoting proliferation and maintaining nuclear morphology in several breast and pancreatic cancer cell lines. Similar effects on cell growth have been evidenced for ZNF414 using in vitro knock-down experiments. Other than this, the function of ZNF414 remains uncharacterized but, as a zinc finger protein with nuclear localization, it is likely to act as a transcription factor. This study aimed at identifying target genes and DNA binding motifs of ZNF414 in pancreatic and breast cancer cells. Methods: We used next generation sequencing methods on Hs700T and MCF-7 cell lines. RNA-seq was used to identify genes regulated by ZNF414 and ChIP-exo and ATAC-seq analyses to uncover its genomic binding regions. Samples for RNA-seq were collected 12h and 24h after transfection with siRNAs targeting ZNF414. For ChIP-exo and ATAC-seq experiments, cells were transfected to transiently overexpress V5-tagged ZNF414. Results: RNA-seq data analyses revealed 33 and 296 differentially expressed genes (DEGs) in Hs700T cells at 12h and 24h time points, respectively. The corresponding amounts of DEGs in MCF-7 cell line were 177 and 556. There were 23 and 108 DEGs in common to both cell lines at 12h and 24h, respectively. Interestingly, gene ontology analyses revealed enrichment of many functional categories related to cellular proliferation in both cell lines, providing a molecular explanation for the observed ZNF414-elicited phenotype. ChIP-exo data was processed using MACE tool and then combined with the ATAC-seq open chromatin signal to identify the most reliable peaks. We looked at the genomic location of these peaks and observed clear enrichment in the promoter and 5'UTR regions, indicating that ZNF414 is a classical transcription factor. Using MEME for de novo motif discovery analyses, we identified several putative binding motifs common to both cell lines, some of which were also found in promoters of DEGs. Conclusion: This study uncovered the transcriptional regulation that underlies the role of ZNF414 as inducer of cellular proliferation in pancreatic and breast cancer cells. Citation Format: Alejandra Rodríguez Martínez, Elisa M. Vuorinen, Anastasia Shcherban, Nina K. Rajala, Matti Nykter, Anne Kallioniemi. ZNF414 as a functionally relevant transcription factor in pancreatic and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3353.

An atlas of chromatin accessibility in the adult human brain.

Most common genetic risk variants associated with neuropsychiatric disease are noncoding and are thought to exert their effects by disrupting the function of cis regulatory elements (CREs), including promoters and enhancers. Within each cell, chromatin is arranged in specific patterns to expose the repertoire of CREs required for optimal spatiotemporal regulation of gene expression. To further understand the complex mechanisms that modulate transcription in the brain, we used frozen postmortem samples to generate the largest human brain and cell-type–specific open chromatin data set to date. Using the Assay for Transposase Accessible Chromatin followed by sequencing (ATAC-seq), we created maps of chromatin accessibility in two cell types (neurons and non-neurons) across 14 distinct brain regions of five individuals. Chromatin structure varies markedly by cell type, with neuronal chromatin displaying higher regional variability than that of non-neurons. Among our findings is an open chromatin region (OCR) specific to neurons of the striatum. When placed in the mouse, a human sequence derived from this OCR recapitulates the cell type and regional expression pattern predicted by our ATAC-seq experiments. Furthermore, differentially accessible chromatin overlaps with the genetic architecture of neuropsychiatric traits and identifies differences in molecular pathways and biological functions. By leveraging transcription factor binding analysis, we identify protein-coding and long noncoding RNAs (lncRNAs) with cell-type and brain region specificity. Our data provide a valuable resource to the research community and we provide this human brain chromatin accessibility atlas as an online database “Brain Open Chromatin Atlas (BOCA)” to facilitate interpretation.

ATACseqQC: a Bioconductor package for post-alignment quality assessment of ATAC-seq data

BackgroundATAC-seq (Assays for Transposase-Accessible Chromatin using sequencing) is a recently developed technique for genome-wide analysis of chromatin accessibility. Compared to earlier methods for assaying chromatin accessibility, ATAC-seq is faster and easier to perform, does not require cross-linking, has higher signal to noise ratio, and can be performed on small cell numbers. However, to ensure a successful ATAC-seq experiment, step-by-step quality assurance processes, including both wet lab quality control and in silico quality assessment, are essential. While several tools have been developed or adopted for assessing read quality, identifying nucleosome occupancy and accessible regions from ATAC-seq data, none of the tools provide a comprehensive set of functionalities for preprocessing and quality assessment of aligned ATAC-seq datasets.ResultsWe have developed a Bioconductor package, ATACseqQC, for easily generating various diagnostic plots to help researchers quickly assess the quality of their ATAC-seq data. In addition, this package contains functions to preprocess aligned ATAC-seq data for subsequent peak calling. Here we demonstrate the utilities of our package using 25 publicly available ATAC-seq datasets from four studies. We also provide guidelines on what the diagnostic plots should look like for an ideal ATAC-seq dataset.ConclusionsThis software package has been used successfully for preprocessing and assessing several in-house and public ATAC-seq datasets. Diagnostic plots generated by this package will facilitate the quality assessment of ATAC-seq data, and help researchers to evaluate their own ATAC-seq experiments as well as select high-quality ATAC-seq datasets from public repositories such as GEO to avoid generating hypotheses or drawing conclusions from low-quality ATAC-seq experiments. The software, source code, and documentation are freely available as a Bioconductor package at https://bioconductor.org/packages/release/bioc/html/ATACseqQC.html.

Correcting nucleotide-specific biases in high-throughput sequencing data

BackgroundHigh-throughput sequence (HTS) data exhibit position-specific nucleotide biases that obscure the intended signal and reduce the effectiveness of these data for downstream analyses. These biases are particularly evident in HTS assays for identifying regulatory regions in DNA (DNase-seq, ChIP-seq, FAIRE-seq, ATAC-seq). Biases may result from many experiment-specific factors, including selectivity of DNA restriction enzymes and fragmentation method, as well as sequencing technology-specific factors, such as choice of adapters/primers and sample amplification methods.ResultsWe present a novel method to detect and correct position-specific nucleotide biases in HTS short read data. Our method calculates read-specific weights based on aligned reads to correct the over- or underrepresentation of position-specific nucleotide subsequences, both within and adjacent to the aligned read, relative to a baseline calculated in assay-specific enriched regions. Using HTS data from a variety of ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq experiments, we show that our weight-adjusted reads reduce the position-specific nucleotide imbalance across reads and improve the utility of these data for downstream analyses, including identification and characterization of open chromatin peaks and transcription-factor binding sites.ConclusionsA general-purpose method to characterize and correct position-specific nucleotide sequence biases fills the need to recognize and deal with, in a systematic manner, binding-site preference for the growing number of HTS-based epigenetic assays. As the breadth and impact of these biases are better understood, the availability of a standard toolkit to correct them will be important.