Recent studies have offered ample insight into genome-wide expression patterns to define pancreatic ductal adenocarcinoma (PDAC) subtypes, although there remains a lack of knowledge regarding the underlying epigenomics of PDAC. Here we perform multi-parametric integrative analyses of chromatin immunoprecipitation-sequencing (ChIP-seq) on multiple histone modifications, RNA-sequencing (RNA-seq), and DNA methylation to define epigenomic landscapes for PDAC subtypes, which can predict their relative aggressiveness and survival. Moreover, we describe the state of promoters, enhancers, super-enhancers, euchromatic, and heterochromatic regions for each subtype. Further analyses indicate that the distinct epigenomic landscapes are regulated by different membrane-to-nucleus pathways. Inactivation of a basal-specific super-enhancer associated pathway reveals the existence of plasticity between subtypes. Thus, our study provides new insight into the epigenetic landscapes associated with the heterogeneity of PDAC, thereby increasing our mechanistic understanding of this disease, as well as offering potential new markers and therapeutic targets.

MiRNAs play a key role in normal physiology and various diseases. miRNA profiling through next generation sequencing (miRNA-seq) has become the main platform for biological research and biomarker discovery. However, analyzing miRNA sequencing data is challenging as it needs significant amount of computational resources and bioinformatics expertise. Several web based analytical tools have been developed but they are limited to processing one or a pair of samples at time and are not suitable for a large scale study. Lack of flexibility and reliability of these web applications are also common issues. We developed a Comprehensive Analysis Pipeline for microRNA Sequencing data (CAP-miRSeq) that integrates read pre-processing, alignment, mature/precursor/novel miRNA detection and quantification, data visualization, variant detection in miRNA coding region, and more flexible differential expression analysis between experimental conditions. According to computational infrastructure, users can install the package locally or deploy it in Amazon Cloud to run samples sequentially or in parallel for a large number of samples for speedy analyses. In either case, summary and expression reports for all samples are generated for easier quality assessment and downstream analyses. Using well characterized data, we demonstrated the pipeline’s superior performances, flexibility, and practical use in research and biomarker discovery. CAP-miRSeq is a powerful and flexible tool for users to process and analyze miRNA-seq data scalable from a few to hundreds of samples. The results are presented in the convenient way for investigators or analysts to conduct further investigation and discovery.

Alcoholic hepatitis (AH) is associated with liver neutrophil infiltration through activated cytokine pathways leading to elevated chemokine expression. Super-enhancers are expansive regulatory elements driving augmented gene expression. Here, we explore the mechanistic role of super-enhancers linking cytokine TNFα with chemokine amplification in AH. RNA-seq and histone modification ChIP-seq of human liver explants show upregulation of multiple CXCL chemokines in AH. Liver sinusoidal endothelial cells (LSEC) are identified as an important source of CXCL expression in human liver, regulated by TNFα/NF-κB signaling. A super-enhancer is identified for multiple CXCL genes by multiple approaches. dCas9-KRAB-mediated epigenome editing or pharmacologic inhibition of Bromodomain and Extraterminal (BET) proteins, transcriptional regulators vital to super-enhancer function, decreases chemokine expression in vitro and decreases neutrophil infiltration in murine models of AH. Our findings highlight the role of super-enhancer in propagating inflammatory signaling by inducing chemokine expression and the therapeutic potential of BET inhibition in AH treatment.

Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-Seq) has been widely used to identify genomic loci of transcription factor (TF) binding and histone modifications. ChIP-Seq data analysis involves multiple steps from read mapping and peak calling to data integration and interpretation. It remains challenging and time-consuming to process large amounts of ChIP-Seq data derived from different antibodies or experimental designs using the same approach. To address this challenge, there is a need for a comprehensive analysis pipeline with flexible settings to accelerate the utilization of this powerful technology in epigenetics research. We have developed a highly integrative pipeline, termed HiChIP for systematic analysis of ChIP-Seq data. HiChIP incorporates several open source software packages selected based on internal assessments and published comparisons. It also includes a set of tools developed in-house. This workflow enables the analysis of both paired-end and single-end ChIP-Seq reads, with or without replicates for the characterization and annotation of both punctate and diffuse binding sites. The main functionality of HiChIP includes: (a) read quality checking; (b) read mapping and filtering; (c) peak calling and peak consistency analysis; and (d) result visualization. In addition, this pipeline contains modules for generating binding profiles over selected genomic features, de novo motif finding from transcription factor (TF) binding sites and functional annotation of peak associated genes. HiChIP is a comprehensive analysis pipeline that can be configured to analyze ChIP-Seq data derived from varying antibodies and experiment designs. Using public ChIP-Seq data we demonstrate that HiChIP is a fast and reliable pipeline for processing large amounts of ChIP-Seq data.

Background: GATK Best Practices workflows are widely used in large-scale sequencing projects and recommend post-alignment processing before variant calling. Two key post-processing steps include the computationally intensive local realignment around known INDELs and base quality score recalibration (BQSR). Both have been shown to reduce erroneous calls; however, the findings are mainly supported by the analytical pipeline that incorporates BWA and GATK UnifiedGenotyper. It is not known whether there is any benefit of post-processing and to what extent the benefit might be for pipelines implementing other methods, especially given that both mappers and callers are typically updated. Moreover, because sequencing platforms are upgraded regularly and the new platforms provide better estimations of read quality scores, the need for post-processing is also unknown. Finally, some regions in the human genome show high sequence divergence from the reference genome; it is unclear whether there is benefit from post-processing in these regions. Results: We used both simulated and NA12878 exome data to comprehensively assess the impact of post-processing for five or six popular mappers together with five callers. Focusing on chromosome 6p21.3, which is a region of high sequence divergence harboring the human leukocyte antigen (HLA) system, we found that local realignment had little or no impact on SNP calling, but increased sensitivity was observed in INDEL calling for the Stampy + GATK UnifiedGenotyper pipeline. No or only a modest effect of local realignment was detected on the three haplotype-based callers and no evidence of effect on Novoalign. BQSR had virtually negligible effect on INDEL calling and generally reduced sensitivity for SNP calling that depended on caller, coverage and level of divergence. Specifically, for SAMtools and FreeBayes calling in the regions with low divergence, BQSR reduced the SNP calling sensitivity but improved the precision when the coverage is insufficient. However, in regions of high divergence (e.g., the HLA region), BQSR reduced the sensitivity of both callers with little gain in precision rate. For the other three callers, BQSR reduced the sensitivity without increasing the precision rate regardless of coverage and divergence level. Conclusions: We demonstrated that the gain from post-processing is not universal; rather, it depends on mapper and caller combination, and the benefit is influenced further by sequencing depth and divergence level. Our analysis highlights the importance of considering these key factors in deciding to apply the computationally intensive post-processing to Illumina exome data.

Epigenetic dysregulation is involved in the etiology and progression of various human diseases. Formalin-fixed paraffin-embedded (FFPE) samples represent the gold standard for archiving pathology samples, and thus FFPE samples are a major resource of samples in clinical research. However, chromatin-based epigenetic assays in the clinical settings are limited to fresh or frozen samples, and are hampered by low chromatin yield in FFPE samples due to the lack of a reliable and efficient chromatin preparation method. Here, we introduce a new chromatin extraction method from FFPE tissues (Chrom-EX PE) for chromatin-based epigenetic assays. During rehydration of FFPE tissues, applying a tissue-level cross-link reversal into the deparaffinized tissue at 65 °C dramatically increased chromatin yield in the soluble fraction. The resulting chromatin is compatible with targeted ChIP-qPCR and genome-wide ChIP-seq approaches. The chromatin prepared by Chrom-EX PE showed a gradual fragmentation pattern with varying incubation temperature. At temperatures below 37 °C, the majority of soluble chromatin is over 1 kb. The soluble chromatin prepared in the range of 45–60 °C showed a typical nucleosomal pattern. And the majority of chromatin prepared at 65 °C is close to mononucleosomal size. These observations indicate that chromatin preparation from FFPE samples can be controlled for downstream chromatin-based epigenetic assays. This study provided a new method that achieves efficient extraction of high-quality chromatin suitable for chromatin-based epigenetic assays with less damage on chromatin. This approach may provide a way to circumvent the over-fixed nature of FFPE tissues for future technology development.