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An improved method for identifying biological circular RNAs

ABSTRACT: Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions.

ORGANISM(S): Mus musculus synthetic construct

PROVIDER: GSE83992 | GEO | 2016/07/05

SECONDARY ACCESSION(S): PRJNA327756

REPOSITORIES: GEO

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An improved method for identifying biological circular RNAs

Project description:Purpose: We are using the illumina sequencing to compare the false positive and true positive circular RNA findings to confine the method to detect the true circular RNAs Methods: The testis whole transcriptome profiling was generated from 4-week mouse testis using illumina Nextseq, duplicated. The sequence reads that passed quality filters were analyzed at the transcript isoform level with TopHat followed by Cufflinks. Results: our data suggest that circular RNAs identified based on junction sequences in the RNA-seq reads, especially those from Illumina Hiseq sequencing, mostly result from template-switching events during reverse transcription by MMLV-derived reverse transcriptases. It is critical to employ reverse transcriptases lacking terminal transferase activity (e.g., MonsterScript) to construct sequencing library or perform RT-PCR for identification and quantification of true circular RNAs. Conclusions: Our study represents the first detailed analysis of retinal transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. The wild type mouse testis RNAs were constructed NGS library by two different enzyme, then parallel sequenced in illumina Nextseq

2016-07-05 | E-GEOD-83992 | biostudies-arrayexpress

Anomalous reverse transcription through chemical modifications in polyadenosine stretches

Project description:Thermostable reverse transcriptases are workhorse enzymes underlying nearly all modern techniques for RNA structure mapping and for transcriptome-wide discovery of RNA chemical modifications. Despite their wide use, these enzymes’ behaviors at chemical modified nucleotides remain poorly understood. Wellington-Oguri et al. recently reported an apparent loss of chemical modification within putatively unstructured polyadenosine stretches modified by dimethyl sulfate or 2’ hydroxyl acylation, as probed by reverse transcription. Here, re-analysis of these and other publicly available data, capillary electrophoresis experiments on chemically modified RNAs, and nuclear magnetic resonance spectroscopy on A 12 and variants show that this effect is unlikely to arise from an unusual structure of polyadenosine. Instead, tests of different reverse transcriptases on chemically modified RNAs and molecules synthesized with single 1-methyladenosines implicate a previously uncharacterized reverse transcriptase behavior: near-quantitative bypass through chemical modifications within polyadenosine stretches. All tested natural and engineered reverse transcriptases (MMLV; SuperScript II, III, and IV; TGIRT-III; and MarathonRT) exhibit this anomalous bypass behavior. Accurate DMS-guided structure modeling of the polyadenylated HIV-1 3´ untranslated region RNA requires taking into account this anomaly. Our results suggest that poly(rA-dT) hybrid duplexes can trigger unexpectedly effective reverse transcriptase bypass and that chemical modifications in poly(A) mRNA tails may be generally undercounted.

2020-04-22 | GSE149061 | GEO

Identification of the Thermoproteus tenax small Rnome

Project description:Small RNAs were isolated from the hyperthermophilic archaeon Thermoproteus tenax and subjected to Illumina Hiseq2000 sequencing. The RNA reads revealed significant abundance of constitutively transcribed CRISPR RNAs and a plethora of C/D box sRNAs. The presence of a rare RNase P RNA variant was verified. Circular RNA molecules could be identified.

2015-09-25 | GSE72127 | GEO

Transcriptomic analysis of the abundance of snoRNAs and their host genes in 4 different cell lines, using low structure bias TGIRT-seq

Project description:Small nucleolar RNAs (snoRNAs) are highly abundant non-coding RNAs whose canonical function is to guide the 2’-O-ribose methylation or pseudouridylation of ribosomal RNAs. They are often encoded in longer genes referred to as their host genes, the expression of which is required for their own biogenesis. Due to their very stable structure, reverse transcription of snoRNAs using standard viral reverse transcriptases is very inefficient, and does not provide an adequate view of the abundance of these small RNAs. We thus employed high-throughput, low structure bias TGIRT-seq sequencing to quantified the abundance of the entire ribodepleted transcriptome, including snoRNAs and their host genes, in 4 different cell lines.

2022-08-10 | GSE209924 | GEO

Homo sapiens

Project description:Circular RNAs deep sequence profiles of human testis

| PRJNA349265 | ENA

Project description:Circular RNAs deep sequence profiles of mouse testis

| PRJNA420752 | ENA

Long- and short-read RNA sequencing from five reproductive organs of boar.

Project description:PacBio SMRTseq long reads and Illumina short reads of pig testis, epididymis, vesicular gland, prostate gland, and bulbourethral gland

2023-08-24 | GSE230506 | GEO

Evolution of a Reverse Transcriptase to Map N1-Methyladenosine in Human mRNA

Project description:Chemical modifications on mRNA are increasingly recognized as a critical regulatory layer of the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile directed evolution platform that rapidly selects for reverse transcriptases that install mutations during reverse transcription at sites of a given type of RNA modification, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. We apply the evolved reverse transcriptase to identify thousands of statistically confident m1A sites in human mRNA, some of which can be detected in antibody-free RNA-seq libraries. Together, this work develops and validates the reverse transcriptase evolution platform and provides new tools, analysis methods, and datasets to study m1A biology.

2019-09-24 | GSE123365 | GEO

N1-methylpseudouridine found within COVID-19 mRNA vaccines produces faithful protein products

Project description:Given the interest in the COVID mRNA vaccines, we sought to investigate how the RNA modification N1-methylpseudouridine (and its related modification, pseudouridine) is read by ribosomes and reverse transcriptases. By looking at reverse transcriptase data, we can gain information on how the modification affects duplex stability, which may have important consequences for the tRNA-mRNA interactions found in the ribosome.

2021-10-25 | GSE186464 | GEO

Identification of small RNA content in Agrobacterium fabrum C58

Project description:Sequencing by the Illumina Genome Analyzer II(36 bp) of the reverse transcribed small RNAs (<500b) using the Illumina directional mRNA-Seq protocol. This was performed on A. fabrum C58 strain from several growth conditions. Reads were then aligned to the genome of the bacteria (Accession number NC_003062, NC_003063, NC_003064 and NC_003065)

2016-03-18 | E-MTAB-2947 | biostudies-arrayexpress

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