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ABSTRACT: Identification and characterization of novel circular RNA

PROVIDER: PRJNA664285 | ENA |

REPOSITORIES: ENA

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Dataset's files

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			Action	DRS
	SRR12667017_1.fastq.gz	Fastqsanger.gz
	SRR12667017_2.fastq.gz	Fastqsanger.gz
	SRR12667018_1.fastq.gz	Fastqsanger.gz
	SRR12667018_2.fastq.gz	Fastqsanger.gz
	SRR12667019_1.fastq.gz	Fastqsanger.gz

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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

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Project description:High-throughput RNA sequencing is employed to quantify transcript abundance and for the de novo identification of novel transcripts, including a family of alternatively spliced RNA molecules called circular RNAs (circRNAs). However, quantification of circRNAs between sequencing libraries remains challenging because of RNA complexity in addition to confounding errors introduced during exonuclease digestion, library preparation and sequencing itself. We have developed a set of synthetic circular RNA spike-ins - termed ‘SynCRS’ - that are directly added to RNA samples before exonuclease digestion and library preparation. These SynCRS are of varying sizes and can be integrated into all NGS workflows and, critically, facilitate the quantitative calibration of circular RNA transcript abundance between samples, tissue types, species and laboratories.

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