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ABSTRACT: Structural basis for template switching by a group II intron-encoded reverse transcriptase

PROVIDER: PRJNA723603 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14301557_1.fastq.gz	Fastqsanger.gz
	SRR14301557_2.fastq.gz	Fastqsanger.gz
	SRR14301558_1.fastq.gz	Fastqsanger.gz
	SRR14301558_2.fastq.gz	Fastqsanger.gz
	SRR14301559_1.fastq.gz	Fastqsanger.gz

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Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

Project description:Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

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Template switching by a group II intron reverse transcriptase: biochemical analysis and implications for RNA-seq

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

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Sequencing artifacts produced by mispriming during reverse transcription in multiple RNA-seq technologies

Project description:The quality of RNA sequencing data relies on specific priming by the primer used for reverse transcription (RT-primer). Non-specific annealing of the RT-primer to the RNA template can generate reads with incorrect cDNA ends and can cause misinterpretation of data (RT mispriming). This kind of artifact in RNA-seq based technologies is underappreciated and currently no adequate tools exist to computationally remove them from published datasets. We show that mispriming can occur with as little as 2 bases of complementarity at the 3' end of the primer followed by intermittent regions of complementarity. We propose an experimental solution to avoid RT-mispriming by performing RNA-seq using thermostable group II intron derived reverse transcriptase (TGIRT-seq).

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BuTT-Seq: a new method for facile profiling of transcription

Project description:A wide range of sequencing methods have been developed to assess nascent RNA transcription and resolve the single-nucleotide position of RNA polymerase genome-wide. These techniques are often burdened with high input material requirements and lengthy protocols. We leveraged the template-switching properties of thermostable group II intron reverse transcriptase (TGIRT) and developed BuTT-Seq (BUlk analysis of nascent Transcript Termini sequencing), which can produce libraries from purified nascent RNA in 6 hours and from as few as 10,000 cells – an improvement of at least 10-fold over existing techniques. BuTT-Seq shows that inhibition of the superelongation complex (SEC) causes promoter-proximal pausing to move upstream in a fashion correlated with subnucleosomal fragments. To address transcriptional regulation in a tissue, BuTT-Seq was used to measure the circadian regulation of transcription from fly heads. All the results indicate that BuTT-Seq is a simple and powerful technique to analyze transcription at a high level of resolution.

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