Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1090626 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28436122_1.fastq.gz | Fastqsanger.gz | |||
| SRR28436122_2.fastq.gz | Fastqsanger.gz | |||
| SRR28436123_1.fastq.gz | Fastqsanger.gz | |||
| SRR28436123_2.fastq.gz | Fastqsanger.gz | |||
| SRR28436124_1.fastq.gz | Fastqsanger.gz |
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Project description:The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.
2019-11-11 | GSE138200 | GEO
Project description:We discovered that PCR-mediated template switching poses a significant challenge in ensemble tagged PCR, particularly in the template sequences with high similarities, which we successfully addressed by introducing a dual barcode. Template switching presents in repair sequencing (repair seq) and severely interfere the interpretation of the association between repair outcomes and the sgRNA in the vicinity. Among the 67 DNA damage response genes, we found that ERCC6L2 was crucial for preventing the DNA end resection.
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Project description:RNAseq experiments of Enterovirus A71 wild type demonstrate that the pyrazine-carboxamide ribonucleotide stimulates catalyzed intra- and intermolecular template switching. These results suggest that pyrazine-2 carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination, but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
2021-09-14 | GSE183959 | GEO
Project description:We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain RNA, and liver FFPE RNA, and demonstrated that chemical capping in conjunction with template switching consistently reduces sequencing bias and improves library quality in comparison with commercially available RNA-seq kits. Finally, we showed the simultaneous detection of miRNAs and mRNAs in FFPE derived samples, underscoring the potential of this workflow for dissecting regulatory networks between miRNAs and their target gene transcripts.
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Project description:Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq
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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
Project description:Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses
| PRJNA762534 | ENA
Project description:Efforts to identify ccRNA by sequencing by gibson circularization after template-switching 5' RACE using a cRNA-end specific primer and subsequent sequencing by Illumina miSeq. Sequencing of wild-type A/WSN/1933 and a variant bearing the mutation T677A in the PB1 subunit.
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Project description:End-to-end chromosome fusions that occur in the context of telomerase deficiency can trigger genomic duplications. These duplications are suggested to arise via Breakage-Fusion-Bridge cycles. To test this hypothesis, we examined end-to-end fusions isolated from C. elegans telomere replication mutants. Genome level rearrangements revealed fused chromosome ends possessing interrupted terminal duplications accompanied by template switching events. These features are very similar to disease-associated duplications of interstitial segments of the human genome. A model termed Fork Stalling and Template Switching has been proposed previously to explain such duplications, where promiscuous replication of large, non-contiguous segments of the genome occurs. Thus, a DNA synthesis-based process can create duplications that seal end-to-end fusions, in the absence of Breakage-Fusion-Bridge cycles.
2011-01-21 | GSE26691 | GEO
Project description:DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.
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