The ENCODE4 long-read RNA-seq collection reveals distinct classes of transcript structure diversity.

The majority of mammalian genes encode multiple transcript isoforms that result from differential promoter use, changes in exonic splicing, and alternative 3' end choice. Detecting and quantifying transcript isoforms across tissues, cell types, and species has been extremely challenging because transcripts are much longer than the short reads normally used for RNA-seq. By contrast, long-read RNA-seq (LR-RNA-seq) gives the complete structure of most transcripts. We sequenced 264 LR-RNA-seq PacBio libraries totaling over 1 billion circular consensus reads (CCS) for 81 unique human and mouse samples. We detect at least one full-length transcript from 87.7% of annotated human protein coding genes and a total of 200,000 full-length transcripts, 40% of which have novel exon junction chains. To capture and compute on the three sources of transcript structure diversity, we introduce a gene and transcript annotation framework that uses triplets representing the transcript start site, exon junction chain, and transcript end site of each transcript. Using triplets in a simplex representation demonstrates how promoter selection, splice pattern, and 3' processing are deployed across human tissues, with nearly half of multi-transcript protein coding genes showing a clear bias toward one of the three diversity mechanisms. Evaluated across samples, the predominantly expressed transcript changes for 74% of protein coding genes. In evolution, the human and mouse transcriptomes are globally similar in types of transcript structure diversity, yet among individual orthologous gene pairs, more than half (57.8%) show substantial differences in mechanism of diversification in matching tissues. This initial large-scale survey of human and mouse long-read transcriptomes provides a foundation for further analyses of alternative transcript usage, and is complemented by short-read and microRNA data on the same samples and by epigenome data elsewhere in the ENCODE4 collection.

New developments on the Encyclopedia of DNA Elements (ENCODE) data portal.

The Encyclopedia of DNA Elements (ENCODE) is an ongoing collaborative research project aimed at identifying all the functional elements in the human and mouse genomes. Data generated by the ENCODE consortium are freely accessible at the ENCODE portal (https://www.encodeproject.org/), which is developed and maintained by the ENCODE Data Coordinating Center (DCC). Since the initial portal release in 2013, the ENCODE DCC has updated the portal to make ENCODE data more findable, accessible, interoperable and reusable. Here, we report on recent updates, including new ENCODE data and assays, ENCODE uniform data processing pipelines, new visualization tools, a dataset cart feature, unrestricted public access to ENCODE data on the cloud (Amazon Web Services open data registry, https://registry.opendata.aws/encode-project/) and more comprehensive tutorials and documentation.

The ENCODE Portal as an Epigenomics Resource.

The Encyclopedia of DNA Elements (ENCODE) web portal hosts genomic data generated by the ENCODE Consortium, Genomics of Gene Regulation, The NIH Roadmap Epigenomics Consortium, and the modENCODE and modERN projects. The goal of the ENCODE project is to build a comprehensive map of the functional elements of the human and mouse genomes. Currently, the portal database stores over 500 TB of raw and processed data from over 15,000 experiments spanning assays that measure gene expression, DNA accessibility, DNA and RNA binding, DNA methylation, and 3D chromatin structure across numerous cell lines, tissue types, and differentiation states with selected genetic and molecular perturbations. The ENCODE portal provides unrestricted access to the aforementioned data and relevant metadata as a service to the scientific community. The metadata model captures the details of the experiments, raw and processed data files, and processing pipelines in human and machine-readable form and enables the user to search for specific data either using a web browser or programmatically via REST API. Furthermore, ENCODE data can be freely visualized or downloaded for additional analyses. © 2019 The Authors. Basic Protocol: Query the portal Support Protocol 1: Batch downloading Support Protocol 2: Using the cart to download files Support Protocol 3: Visualize data Alternate Protocol: Query building and programmatic access.

Neural substrates of resisting craving during cigarette cue exposure.

BACKGROUND: In cigarette smokers, the most commonly reported areas of brain activation during visual cigarette cue exposure are the prefrontal, anterior cingulate, and visual cortices. We sought to determine changes in brain activity in response to cigarette cues when smokers actively resist craving. METHODS: Forty-two tobacco-dependent smokers underwent functional magnetic resonance imaging, during which they were presented with videotaped cues. Three cue presentation conditions were tested: cigarette cues with subjects allowing themselves to crave (cigarette cue crave), cigarette cues with the instruction to resist craving (cigarette cue resist), and matched neutral cues. RESULTS: Activation was found in the cigarette cue resist (compared with the cigarette cue crave) condition in the left dorsal anterior cingulate cortex (ACC), posterior cingulate cortex (PCC), and precuneus. Lower magnetic resonance signal for the cigarette cue resist condition was found in the cuneus bilaterally, left lateral occipital gyrus, and right postcentral gyrus. These relative activations and deactivations were more robust when the cigarette cue resist condition was compared with the neutral cue condition. CONCLUSIONS: Suppressing craving during cigarette cue exposure involves activation of limbic (and related) brain regions and deactivation of primary sensory and motor cortices.

Cigarette smoking saturates brain alpha 4 beta 2 nicotinic acetylcholine receptors.

CONTEXT: 2-[18F]fluoro-3-(2(S)-azetidinylmethoxy) pyridine (2-F-A-85380, abbreviated as 2-FA) is a recently developed radioligand that allows for visualization of brain alpha 4 beta 2* nicotinic acetylcholine receptors (nAChRs) with positron emission tomography (PET) scanning in humans. OBJECTIVE: To determine the effect of cigarette smoking on alpha 4 beta 2* nAChR occupancy in tobacco-dependent smokers. DESIGN: Fourteen 2-FA PET scanning sessions were performed. During the PET scanning sessions, subjects smoked 1 of 5 amounts (none, 1 puff, 3 puffs, 1 full cigarette, or to satiety [2(1/2) to 3 cigarettes]). SETTING: Academic brain imaging center. PARTICIPANTS: Eleven tobacco-dependent smokers (paid volunteers). Main Outcome Measure Dose-dependent effect of smoking on occupancy of alpha 4 beta 2* nAChRs, as measured with 2-FA and PET in nAChR-rich brain regions. RESULTS: Smoking 0.13 (1 to 2 puffs) of a cigarette resulted in 50% occupancy of alpha 4 beta 2* nAChRs for 3.1 hours after smoking. Smoking a full cigarette (or more) resulted in more than 88% receptor occupancy and was accompanied by a reduction in cigarette craving. A venous plasma nicotine concentration of 0.87 ng/mL (roughly 1/25th of the level achieved in typical daily smokers) was associated with 50% occupancy of alpha 4 beta 2* nAChRs. CONCLUSIONS: Cigarette smoking in amounts used by typical daily smokers leads to nearly complete occupancy of alpha 4 beta 2* nAChRs, indicating that tobacco-dependent smokers maintain alpha 4 beta 2* nAChR saturation throughout the day. Because prolonged binding of nicotine to alpha 4 beta 2* nAChRs is associated with desensitization of these receptors, the extent of receptor occupancy found herein suggests that smoking may lead to withdrawal alleviation by maintaining nAChRs in the desensitized state.