scholarly journals Sex-specific transcriptomic and epitranscriptomic signatures of PTSD-like fear acquisition.

2021 ◽  
Author(s):  
Andre Martins Reis ◽  
Jillian Hammond ◽  
Igor Stevanovski ◽  
Jonathon Arnold ◽  
Iain McGregor ◽  
...  
Keyword(s):  
Stress Disorder ◽  
Transcriptional Response ◽  
Rna Seq ◽  
Rna Modifications ◽  
Oxford Nanopore ◽  
Recent Emergence ◽  
Mrna Transcripts ◽  
Unbiased Gene ◽  
Future Work ◽  
Central Tool

Our understanding of the molecular pathology of posttraumatic stress disorder (PTSD) is rapidly evolving and is being driven by advances in sequencing techniques. Conventional short-read RNA sequencing (RNA-seq) is a central tool in transcriptomics research that enables unbiased gene expression profiling. With the recent emergence of Oxford Nanopore direct RNA-seq (dRNA-seq), it is now also possible to interrogate diverse RNA modifications, collectively known as the epitranscriptome. Here, we present our analyses of the male and female mouse amygdala transcriptome and epitranscriptome, obtained using parallel Illumina RNA-seq and Oxford Nanopore dRNA-seq, associated with the acquisition of PTSD-like fear induced by Pavlovian cued-fear conditioning. We report significant sex-specific differences in the amygdala transcriptional response during fear acquisition, and a range of shared and dimorphic epitranscriptomic signatures. Differential RNA modifications are enriched among mRNA transcripts associated with neurotransmitter regulation and mitochondrial function, many of which have been previously implicated in PTSD. Very few differentially modified transcripts are also differentially expressed, suggesting an influential, expression-independent role for epitranscriptional regulation in PTSD-like fear-acquisition. Overall, our application of conventional and newly developed methods provides a platform for future work that will lead to new insights into and therapeutics for PTSD.

scholarly journals NERD-seq: A novel approach of Nanopore direct RNA sequencing that expands representation of non-coding RNAs

2021 ◽  
Author(s):  
Luke Saville ◽  
Yubo Cheng ◽  
Babita Gollen ◽  
Liam Mitchell ◽  
Matthew Stuart-Edwards ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Standard Approach ◽  
Rna Seq ◽  
Rna Modifications ◽  
Current Standard ◽  
Base Calling ◽  
Novel Approach ◽  
Oxford Nanopore ◽  
Non Coding Rnas ◽  
Sequencing Platforms

The new next-generation sequencing platforms by Oxford Nanopore Technologies for direct RNA sequencing (direct RNA-seq) allow for an in-depth and comprehensive study of the epitranscriptome by enabling direct base calling of RNA modifications. Non-coding RNAs constitute the most frequently documented targets for RNA modifications. However, the current standard direct RNA-seq approach is unable to detect many of these RNAs. Here we present NERD-seq, a sequencing approach which enables the detection of multiple classes of non-coding RNAs excluded by the current standard approach. Using total RNA from a tissue with high known transcriptional and non-coding RNA activity in mouse, the brain hippocampus, we show that, in addition to detecting polyadenylated coding and non-coding transcripts as the standard approach does, NERD-seq is able to significantly expand the representation for other classes of RNAs such as snoRNAs, snRNAs, scRNAs, srpRNAs, tRNAs, rRFs and non-coding RNAs originating from LINE L1 elements. Thus, NERD-seq presents a new comprehensive direct RNA-seq approach for the study of epitranscriptomes in brain tissues and beyond.

scholarly journals The assembled and annotated genome of the pigeon louse Columbicola columbae, a model ectoparasite

2021 ◽  
Vol 11 (2) ◽  
Author(s):  
James G Baldwin-Brown ◽  
Scott M Villa ◽  
Anna I Vickrey ◽  
Kevin P Johnson ◽  
Sarah E Bush ◽  
...  
Keyword(s):  
Low Cost ◽  
Single Copy ◽  
Odorant Receptors ◽  
Rna Seq ◽  
Pediculus Humanus ◽  
Final Assembly ◽  
Oxford Nanopore ◽  
Genes Encoding ◽  
Host Parasite ◽  
G Protein Coupled

Abstract The pigeon louse Columbicola columbae is a longstanding and important model for studies of ectoparasitism and host-parasite coevolution. However, a deeper understanding of its evolution and capacity for rapid adaptation is limited by a lack of genomic resources. Here, we present a high-quality draft assembly of the C. columbae genome, produced using a combination of Oxford Nanopore, Illumina, and Hi-C technologies. The final assembly is 208 Mb in length, with 12 chromosome-size scaffolds representing 98.1% of the assembly. For gene model prediction, we used a novel clustering method (wavy_choose) for Oxford Nanopore RNA-seq reads to feed into the MAKER annotation pipeline. High recovery of conserved single-copy orthologs (BUSCOs) suggests that our assembly and annotation are both highly complete and highly accurate. Consistent with the results of the only other assembled louse genome, Pediculus humanus, we find that C. columbae has a relatively low density of repetitive elements, the majority of which are DNA transposons. Also similar to P. humanus, we find a reduced number of genes encoding opsins, G protein-coupled receptors, odorant receptors, insulin signaling pathway components, and detoxification proteins in the C. columbae genome, relative to other insects. We propose that such losses might characterize the genomes of obligate, permanent ectoparasites with predictable habitats, limited foraging complexity, and simple dietary regimes. The sequencing and analysis for this genome were relatively low cost, and took advantage of a new clustering technique for Oxford Nanopore RNAseq reads that will be useful to future genome projects.

scholarly journals RNA Modification Level Estimation with pulseR

Genes ◽  
2018 ◽  
Vol 9 (12) ◽  
pp. 619
Author(s):  
Etienne Boileau ◽  
Christoph Dieterich
Keyword(s):  
Experimental Approach ◽  
High Efficiency ◽  
Rna Modification ◽  
Model Parameters ◽  
Metabolic Labeling ◽  
Rna Seq ◽  
Rna Modifications ◽  
Log Odds ◽  
Pros And Cons ◽  
Wide Scale

RNA modifications regulate the complex life of transcripts. An experimental approach called LAIC-seq was developed to characterize modification levels on a transcriptome-wide scale. In this method, the modified and unmodified molecules are separated using antibodies specific for a given RNA modification (e.g., m6A). In essence, the procedure of biochemical separation yields three fractions: Input, eluate, and supernatent, which are subjected to RNA-seq. In this work, we present a bioinformatics workflow, which starts from RNA-seq data to infer gene-specific modification levels by a statistical model on a transcriptome-wide scale. Our workflow centers around the pulseR package, which was originally developed for the analysis of metabolic labeling experiments. We demonstrate how to analyze data without external normalization (i.e., in the absence of spike-ins), given high efficiency of separation, and how, alternatively, scaling factors can be derived from unmodified spike-ins. Importantly, our workflow provides an estimate of uncertainty of modification levels in terms of confidence intervals for model parameters, such as gene expression and RNA modification levels. We also compare alternative model parametrizations, log-odds, or the proportion of the modified molecules and discuss the pros and cons of each representation. In summary, our workflow is a versatile approach to RNA modification level estimation, which is open to any read-count-based experimental approach.

scholarly journals Decoding the Epitranscriptional Landscape from Native RNA Sequences

2018 ◽  
Author(s):  
Thidathip Wongsurawat ◽  
Piroon Jenjaroenpun ◽  
Trudy M. Wassenaar ◽  
Taylor D Wadley ◽  
Visanu Wanchai ◽  
...  
Keyword(s):  
Growth Conditions ◽  
Untranslated Regions ◽  
Metabolic Adaptation ◽  
Rna Modifications ◽  
Rna Sequences ◽  
Rna Methylation ◽  
E Coli ◽  
Oxford Nanopore ◽  
G Quadruplex ◽  
Bioinformatic Tool

AbstractSequencing of native RNA and corresponding cDNA was performed using Oxford Nanopore Technology. The % Error of Specific Bases (%ESB) was higher for native RNA than for cDNA, which enabled detection of ribonucleotide modification sites. Based on %ESB differences of the two templates, a bioinformatic tool ELIGOS was developed and applied to rRNAs of E. coli, yeast and human cells. ELIGOS captured 91%, 95%, ∼75%, respectively, of the known variety of RNA methylation sites in these rRNAs. Yeast transcriptomes from different growth conditions were also compared, which identified an association between metabolic adaptation and inferred RNA modifications. ELIGOS was further applied to human transcriptome datasets, which identified the well-known DRACH motif containing N6-methyadenine being located close to 3’-untranslated regions of mRNA. Moreover, the RNA G-quadruplex motif was uncovered by ELIGOS. In summary, we have developed an experimental method coupled with bioinformatic software to uncover native RNA modifications and secondary-structures within transcripts.

scholarly journals Role of the cytoskeleton in muscle transcriptional responses to altered use

2013 ◽  
Vol 45 (8) ◽  
pp. 321-331 ◽  
Author(s):  
Gretchen A. Meyer ◽  
Simon Schenk ◽  
Richard L. Lieber
Keyword(s):  
Mechanical Response ◽  
Fiber Type ◽  
Transcriptional Response ◽  
Primary Component ◽  
Transcriptional Responses ◽  
Expression Of Genes ◽  
Elevated Expression ◽  
Transcriptional Changes ◽  
Type Shift ◽  
Future Work

In this work, the interaction between the loss of a primary component of the skeletal muscle cytoskeleton, desmin, and two common physiological stressors, acute mechanical injury and aging, were investigated at the transcriptional, protein, and whole muscle levels. The transcriptional response of desmin knockout ( des −/−) plantarflexors to a bout of 50 eccentric contractions (ECCs) showed substantial overlap with the response in wild-type ( wt) muscle. However, changes in the expression of genes involved in muscle response to injury were blunted in adult des −/− muscle compared with wt (fold change with ECC in des −/− and wt, respectively: Mybph, 1.4 and 2.9; Xirp1, 2.2 and 5.7; Csrp3, 1.8 and 4.3), similar to the observed blunted mechanical response (torque drop: des −/− 30.3% and wt 55.5%). Interestingly, in the absence of stressors, des −/− muscle exhibited elevated expression of many these genes compared with wt. The largest transcriptional changes were observed in the interaction between aging and the absence of desmin, including many genes related to slow fiber pathway (Myh7, Myl3, Atp2a2, and Casq2) and insulin sensitivity (Tlr4, Trib3, Pdk3, and Pdk4). Consistent with these transcriptional changes, adult des −/− muscle exhibited a significant fiber type shift from fast to slow isoforms of myosin heavy chain ( wt, 5.3% IIa and 71.7% IIb; des −/−, 8.4% IIa and 61.4% IIb) and a decreased insulin-stimulated glucose uptake ( wt, 0.188 μmol/g muscle/20 min; des −/−, 0.085 μmol/g muscle/20 min). This work points to novel areas of influence of this cytoskeletal protein and directs future work to elucidate its function.

Next-generation RNA sequencing reveals transcriptomic changes after brief exposure to preoperative nab-paclitaxel, bevacizumab, and trastuzumab.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. 10508-10508
Author(s):  
Vinay Varadan ◽  
Sitharthan Kamalakaran ◽  
Angel Janevski ◽  
Nila Banerjee ◽  
Kimberly Lezon-Geyda ◽  
...  
Keyword(s):  
Multiple Testing ◽  
Day Treatment ◽  
Transcriptional Response ◽  
Enrichment Analysis ◽  
Transcript Abundance ◽  
Differentially Expressed ◽  
Read Length ◽  
Breast Cancers ◽  
Rna Seq ◽  
Multiple Testing Correction

10508 Background: Identification of differentially expressed transcripts after brief exposure to preoperative therapy can help determine likely response markers. We quantify and compare differential gene and isoform expression using RNA-seq on patient samples with 10 day exposure to one dose of trastuzumab, bevacizumab or nab-paclitaxel. Methods: We sequenced transcriptomes of 23 pairs of core biopsy RNA from breast cancers pre/post 10 day exposure to therapy. Paired-end sequencing was done on the Illumina GAII platform using amplified total RNA with 74bp read length, yielding data on transcript abundance for a total of 22,160 genes and 34,449 transcripts. Differential expression of transcripts between pre/post samples was estimated assuming Poisson-distributed read-counts, followed by multiple testing correction and enrichment analysis of 185 KEGG pathways. Results: PAM50-based clustering showed individual samples cluster together, demonstrating that tumor subtypes do not change over the 10-day treatment (SABCS 2011). We identified genes that were significantly differentially expressed (p<0.05; FDR<0.1) in at least 60% of samples within each therapy arm: 780 genes in trastuzumab, 302 in bevacizumab, and 176 in nab-paclitaxel. Surprisingly, only THAP11 and TINF2 were common amongst them. THAP11 is involved in stem cell maintenance and TINF2 is important for regulation of telomere length. Immune system and metabolism-related pathways were commonly affected (p<0.05) across all arms. The bevacizumab arm showed significant down-regulation of angiogenesis-associated genes: ESM1 and VEGFR2 in > 80% of samples. The nab-paclitaxel arm exhibited changes in TGF-beta signaling, Nod-like receptor and Wnt signaling. The trastuzumab arm exhibited consistent alteration of ErbB2 and mTOR pathways, with SOX11 and TOP2B downregulated in every sample. Conclusions: This is the first study to compare gene expression with brief exposure across therapies using RNA-seq technology. The unique aspects of transcriptional response to each treatment underscore the need for specific markers of therapeutic response to nab-paclitaxel, bevacizumab and trastuzumab.

RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae

2016 ◽  
Vol 16 (6) ◽  
pp. 657-669 ◽  
Author(s):  
Mehmet Tardu ◽  
Ugur Meric Dikbas ◽  
Ibrahim Baris ◽  
Ibrahim Halil Kavakli
Keyword(s):  
Red Light ◽  
Transcriptional Response ◽  
Red Alga ◽  
Cyanidioschyzon Merolae ◽  
Rna Seq

scholarly journals Poly(A)-ClickSeq: click-chemistry for next-generation 3´-end sequencing without RNA enrichment or fragmentation

2017 ◽  
Author(s):  
Andrew Routh ◽  
Ping Ji ◽  
Elizabeth Jaworski ◽  
Zheng Xia ◽  
Wei Li ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Simple Procedure ◽  
Alternative Polyadenylation ◽  
Rna Seq ◽  
Cdna Synthesis ◽  
Novel Approach ◽  
Genome Wide ◽  
Recent Emergence ◽  
Complex Sample ◽  
A Site

AbstractThe recent emergence of alternative polyadenylation (APA) as an engine driving transcriptomic diversity has stimulated the development of sequencing methodologies designed to assess genome-wide polyadenylation events. The goal of these approaches is to enrich, partition, capture, and ultimately sequence poly(A) site junctions. However, these methods often require poly(A) enrichment, 3´ linker ligation steps, and RNA fragmentation, which can necessitate higher levels of starting RNA, increase experimental error, and potentially introduce bias. We recently reported a click-chemistry based method for generating RNAseq libraries called “ClickSeq”. Here, we adapt this method to direct the cDNA synthesis specifically toward the 3´ UTR/poly(A) tail junction of cellular RNA. With this novel approach, we demonstrate sensitive and specific enrichment for poly(A) site junctions without the need for complex sample preparation, fragmentation or purification. Poly(A)-ClickSeq (PAC-seq) is therefore a simple procedure that generates high-quality RNA-seq poly(A) libraries. As a proof-of-principle, we utilized PAC-seq to explore the poly(A) landscape of both human and Drosophila cells in culture and observed outstanding overlap with existing poly(A) databases and also identified previously unannotated poly(A) sites. Moreover, we utilize PAC-seq to quantify and analyze APA events regulated by CFIm25 illustrating how this technology can be harnessed to identify alternatively polyadenylated RNA.

scholarly journals Graphmap2 - splice-aware RNA-seq mapper for long reads

2019 ◽  
Author(s):  
Josip Marić ◽  
Ivan Sović ◽  
Krešimir Križanović ◽  
Niranjan Nagarajan ◽  
Mile Šikić
Keyword(s):  
State Of The Art ◽  
The State ◽  
Rna Seq ◽  
Link Type ◽  
Pacific Biosciences ◽  
Long Reads ◽  
Oxford Nanopore

AbstractIn this paper we present Graphmap2, a splice-aware mapper built on our previously developed DNA mapper Graphmap. Graphmap2 is tailored for long reads produced by Pacific Biosciences and Oxford Nanopore devices. It uses several newly developed algorithms which enable higher precision and recall of correctly detected transcripts and exon boundaries. We compared its performance with the state-of-the-art tools Minimap2 and Gmap. On both simulated and real datasets Graphmap2 achieves higher mappability and more correctly recognized exons and their ends. In addition we present an analysis of potential of splice aware mappers and long reads for the identification of previously unknown isoforms and even genes. The Graphmap2 tool is publicly available at https://github.com/lbcb-sci/graphmap2.

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