Reconstruction of circular RNAs using Illumina and Nanopore RNA-seq datasets

AbstractCircular RNAs (circRNAs) are involved in various biological processes and in disease pathogenesis. However, only a small number of functional circRNAs have been identified among hundreds of thousands of circRNA species, partly because most current methods are based on circular junction counts and overlook the fact that circRNA is formed from the host gene by back-splicing (BS). To distinguish between expression originating from BS and that from the host gene, we present DEBKS, a software program to streamline the discovery of differential BS between two rRNA-depleted RNA sequencing (RNA-seq) sample groups. By applying real and simulated data and employing RT-qPCR for validation, we demonstrate that DEBKS is efficient and accurate in detecting circRNAs with differential BS events between paired and unpaired sample groups. DEBKS is available at https://github.com/yangence/DEBKS as open-source software.

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Identification and Characterization of Circular RNAs in Longissimus Dorsi Muscle Tissue from Two Goat Breeds using RNA-Seq

10.21203/rs.3.rs-944970/v1 ◽

2021 ◽

Author(s):

Jiyuan Shen ◽

Huimin Zhen ◽

Lu Li ◽

Yuting Zhang ◽

Jiqing Wang ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Fiber ◽

Muscle Development ◽

Production Performance ◽

Differentially Expressed ◽

Circular Rnas ◽

Longissimus Dorsi ◽

Rna Seq ◽

Skeletal Muscle Development ◽

Fiber Size

Abstract Background: Circular RNAs (circRNAs) are a class of non-coding RNA that play crucial roles in the development of skeletal muscle. However, little is known about the role of circRNAs in caprine skeletal muscle. In this study, the muscle fiber size and expression profiles of circRNAs were compared in Longissimus dorsi muscle of Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with significant phenotypic differences in meat production performance, using hematoxylin and eosin staining and RNA-Seq, respectively.Results: The muscle fiber size in LC goats were larger than those in ZB goats (P < 0.05). A total of 10,875 circRNAs were identified and 214 of these were differentially expressed between the two caprine breeds. The authentication and expression levels of 20 circRNAs were confirmed using reverse transcriptase-polymerase chain reaction (RT-PCR) and DNA sequencing. The parent genes of differentially expressed circRNAs were mainly enriched in connective tissue development, Rap1, cGMP-PKG, cAMP and Ras signaling pathway. Some miRNAs reportedly associated with skeletal muscle development and intramuscular fat deposition would be targeted by several differentially expressed circRNAs and the most highly expressed circRNA (circ_001086).Conclusion: These results provide an improved understanding of the functions of circRNAs in skeletal muscle development of goats.

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Global accumulation of circRNAs during aging in Caenorhabditis elegans

10.1101/175026 ◽

2017 ◽

Author(s):

Mariela Cortés-López ◽

Matthew Gruner ◽

Daphne A. Cooper ◽

Hannah N. Gruner ◽

Alexandru-Ioan Voda ◽

...

Keyword(s):

Global Scale ◽

Circular Rnas ◽

Rna Seq ◽

Aging Research ◽

C Elegans ◽

Fourth Larval Stage ◽

Functional Consequences ◽

Exceptional Stability ◽

Massive Accumulation

SummaryCircular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5´ and 3´ ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated 1,166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by qRT-PCR. The age-accumulation of circRNAs was not accompanied by increased expression of linear RNAs from the same host genes. We attribute the global scale of circRNA age-accumulation to the high composition of postmitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine if there are functional consequences of circRNA accumulation during aging.

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RNA-Seq Profiling of Circular RNAs and the Oncogenic Role of circPVT1 in Cutaneous Squamous Cell Carcinoma

OncoTargets and Therapy ◽

10.2147/ott.s252233 ◽

2020 ◽

Vol Volume 13 ◽

pp. 6777-6788

Author(s):

Shuang Chen ◽

Junli Ding ◽

Yunlin Wang ◽

Tao Lu ◽

Lili Wang ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Cell Carcinoma ◽

Squamous Cell ◽

Cutaneous Squamous Cell Carcinoma ◽

Circular Rnas ◽

Rna Seq

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P5398CircRNAs deregulation in heart failure patients

European Heart Journal ◽

10.1093/eurheartj/ehz746.0358 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

S Greco ◽

A Made' ◽

M Longo ◽

R Tikhomirov ◽

S Castelvecchio ◽

...

Keyword(s):

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Functional Characterization ◽

Enrichment Analysis ◽

Host Gene ◽

Circular Rnas ◽

Amplification Efficiency ◽

Rna Seq ◽

Bioinformatics Pipeline

Abstract Background Circular RNAs (circRNAs) are an emerging class of noncoding RNAs stemming from the splicing and circularization of pre-mRNAs exons. CircRNAs can regulate transcription and splicing, sequester microRNAs acting as “sponge” and inducing the respective targets, and bind to RNA binding proteins. Recently, they have been found deregulated in dilated cardiomyopathies (DCM), one of the cardiovascular diseases with the worst rate of morbidity and mortality, and whose molecular mechanisms are only partially known. Purpose Therein, we will evaluate in ischemic DCM patients the modulation of 17 circRNAs, 14 out of them obtained from literature data on DCM ischemic or not, while the other 3 were circRNAs not characterized in the heart previously. The study aims to identify circRNAs candidates for further functional characterization in DCM. In addition, as differential expression (DE) analysis is not easily performed for circRNAs in RNA-seq datasets, the validated circRNAs will be used to set up the most specific and sensitive bioinformatics pipeline for circRNA-DE analysis. Methods We designed divergent and convergent specific primers for 17 circRNAs and their host gene, respectively, and their amplification efficiency was measured by RT-qPCR. Transcripts expression was measured in left ventricle biopsies of 12 patients affected by non end-stage ischemic HF and of 12 matched controls. Results We identified cPVT1, cANKRD17, cBPTF as DE, and validated the modulation of 5 out of the 14 DCM-related circRNAs (cHIPK3, cALPK2, cPCMTD1, cNEBL, cSLC8A1), while cPDRM5, cTTN1 showed opposite modulation, which may be due to the specific disease condition. All of them were modulated differently from the respective host gene. CircRNA/miRNA interactions were predicted using Starbase 3.0. Next, mRNAs-targets of the identified miRNAs were predicted by mirDIP 4.1 and intersected with gene expression datasets of the same patients, previously obtained by microarray analysis. We found that cBPTF and cANKRD17 might sponge 12 and 2 miRNAs, respectively. Enrichment analysis of the relevant targets identified several important pathways implicated in DCM, such as MAPK, FoxO, EGFR, VEGF and Insulin/IGF pathways. In addition, deep RNA-Seq analysis that is currently ongoing and the validated circRNAs will be used to optimize the bioinformatics pipeline for circRNA DE analysis. Conclusions We identified a subset of circRNAs deregulated in ischemic HF potentially implicated in HF pathogenesis.

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The Landscape of MicroRNA, Piwi-Interacting RNA, and Circular RNA in Human Saliva

Clinical Chemistry ◽

10.1373/clinchem.2014.230433 ◽

2015 ◽

Vol 61 (1) ◽

pp. 221-230 ◽

Cited By ~ 318

Author(s):

Jae Hoon Bahn ◽

Qing Zhang ◽

Feng Li ◽

Tak-Ming Chan ◽

Xianzhi Lin ◽

...

Keyword(s):

Body Fluid ◽

Expression Profiles ◽

Embryonic Stem ◽

Bioinformatic Analysis ◽

Circular Rna ◽

Human Saliva ◽

Body Fluids ◽

Individual Variability ◽

Circular Rnas ◽

Rna Seq

Abstract BACKGROUND Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

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RNA-Seq Profiling of Circular RNAs During Development of Hindgut in Rat Embryos With Ethylenethiourea-Induced Anorectal Malformations

Frontiers in Genetics ◽

10.3389/fgene.2021.605015 ◽

2021 ◽

Vol 12 ◽

Author(s):

Si Ying Li ◽

Chen Yi Wang ◽

Yun Xia Xiao ◽

Xiao Bing Tang ◽

Zheng Wei Yuan ◽

...

Keyword(s):

Molecular Mechanisms ◽

Transforming Growth Factor ◽

Quantitative Polymerase Chain Reaction ◽

Anorectal Malformations ◽

Normal Group ◽

Differentially Expressed ◽

Mitogen Activated Protein Kinase ◽

Circular Rnas ◽

Rna Seq ◽

Pregnant Rats

Anorectal malformations (ARMs) are among the most common congenital terminal digestive tract malformations. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play roles in the development of the digestive system; however, their contributions to the pathogenesis of ARMs are not well-established. In this study, we explored the mechanism underlying ethylenethiourea (ETU)-induced ARMs by profiling circRNA expression via RNA-seq and constructing a regulatory circRNA-miRNA-mRNA network. Nine pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (ARM group) on gestational day 10 (GD10), and another 9 pregnant rats received a similar dose of saline (normal group) as a control. Embryos were obtained by cesarean section on the key time-points of anorectal development (GD14, GD15, and GD16). Hindgut samples isolated from the fetuses were evaluated by high-throughput sequencing and differentially expressed circRNAs were validated by reverse transcription-quantitative polymerase chain reaction, agarose gel electrophoresis, and Sanger cloning and sequencing. A total of 18295 circRNAs were identified in the normal and ARM groups. Based on the 425 differentially expressed circRNAs (|Fc| > 2, p < 0.05), circRNA-miRNA and miRNA-mRNA pairs were predicted using miREAP, miRanda, and TargetScan. A total of 55 circRNAs (14 up- and 41 downregulated in the ARM group compared to the normal group) were predicted to bind to 195 miRNAs and 947 mRNAs. Competing endogenous RNA networks and a Kyoto Encyclopedia of Genes and Genomes analysis revealed that novel_circ_001042 had the greatest connectivity and was closely related to ARM-associated signaling pathways, such as the Wingless Type MMTV integration site family, mitogen-activated protein kinase, and transforming growth factor-β pathways. These results provide original insight into the roles of circRNAs in ARMs and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.

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Sensitive, reliable, and robust circRNA detection from RNA-seq with CirComPara2

10.1101/2021.02.18.431705 ◽

2021 ◽

Author(s):

Enrico Gaffo ◽

Alessia Buratin ◽

Anna Dal Molin ◽

Stefania Bortoluzzi

Keyword(s):

Real Data ◽

Detection Algorithm ◽

Detection Methods ◽

Circular Rnas ◽

Data Sets ◽

Rna Seq ◽

Bioinformatics Tool ◽

Diverse Data ◽

High Throughput Study ◽

Discovery Rates

AbstractCurrent methods for identifying circular RNAs (circRNAs) suffer from low discovery rates and inconsistent performance in diverse data sets. Therefore, the applied detection algorithm can bias high-throughput study findings by missing relevant circRNAs. Here, we show that our bioinformatics tool CirComPara2 (https://github.com/egaffo/CirComPara2), by combining multiple circRNA detection methods, consistently achieves high recall rates without loss of precision in simulated and different real-data sets.

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A CLEAR pipeline for direct comparison of circular and linear RNA expression

10.1101/668657 ◽

2019 ◽

Author(s):

Xu-Kai Ma ◽

Meng-Ran Wang ◽

Chu-Xiao Liu ◽

Rui Dong ◽

Gordon G. Carmichael ◽

...

Keyword(s):

Ribosomal Rna ◽

Circular Rnas ◽

Rna Expression ◽

Computational Pipeline ◽

Rna Seq ◽

Link Type ◽

Genome Wide ◽

A Genome

ABSTRACTSequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with sequences from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, but a direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. This is because quantification of BSJ fragments differs from that of linear RNA expression that uses normalized RNA-seq fragments mapped to the whole gene bodies. Here, we have developed a computational pipeline for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CLEAR, https://github.com/YangLab/CLEAR). A new quantitation parameter, FPB (fragments per billion mapped bases), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Then, circular and linear RNA expression are directly compared by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression using linear RNA expression as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be identified for further investigation.

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Recent Applications of RNA Sequencing in Food and Agriculture

10.5772/intechopen.97500 ◽

2021 ◽

Author(s):

Venkateswara R. Sripathi ◽

Varsha C. Anche ◽

Zachary B. Gossett ◽

Lloyd T. Walker

Keyword(s):

Rna Sequencing ◽

Alternative Polyadenylation ◽

Cost Effective ◽

Circular Rnas ◽

Rna Seq ◽

Complete Collection ◽

Specific Expression ◽

Food And Agriculture ◽

Allele Specific ◽

Next Generation Sequencing Ngs

RNA sequencing (RNA-Seq) is the leading, routine, high-throughput, and cost-effective next-generation sequencing (NGS) approach for mapping and quantifying transcriptomes, and determining the transcriptional structure. The transcriptome is a complete collection of transcripts found in a cell or tissue or organism at a given time point or specific developmental or environmental or physiological condition. The emergence and evolution of RNA-Seq chemistries have changed the landscape and the pace of transcriptome research in life sciences over a decade. This chapter introduces RNA-Seq and surveys its recent food and agriculture applications, ranging from differential gene expression, variants calling and detection, allele-specific expression, alternative splicing, alternative polyadenylation site usage, microRNA profiling, circular RNAs, single-cell RNA-Seq, metatranscriptomics, and systems biology. A few popular RNA-Seq databases and analysis tools are also presented for each application. We began to witness the broader impacts of RNA-Seq in addressing complex biological questions in food and agriculture.

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