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CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing

ABSTRACT: CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing

PROVIDER: PRJEB62851 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR11523783.fastq.gz	Fastqsanger.gz
	ERR11523784.fastq.gz	Fastqsanger.gz
	ERR11523785.fastq.gz	Fastqsanger.gz
	ERR11523786.fastq.gz	Fastqsanger.gz
	ERR11523787.fastq.gz	Fastqsanger.gz

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CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing

Project description:Here we describe CapTrap-Seq, an experimental workflow designed to address the problem of reduced transcript end detection by long-read RNA sequencing methods, especially at the 5' ends. We apply CapTrap-Seq to profile transcriptomes of the human heart and brain and we compared the obtained results with other library preparation approaches. CapTrap-Seq is a platform-agnostic method and here tested the method by using 3 different long-read sequencing platforms: MinION (ONT), Sequel (PacBaio) and Sequel II (PacBio).

2023-12-20 | E-MTAB-13063 | biostudies-arrayexpress

Benchmarking full-length transcript single cell mRNA sequencing protocols.

Project description:BackgroundSingle cell mRNA sequencing technologies have transformed our understanding of cellular heterogeneity and identity. For sensitive discovery or clinical marker estimation where high transcript capture per cell is needed only plate-based techniques currently offer sufficient resolution.ResultsHere, we present a performance evaluation of four different plate-based scRNA-seq protocols. Our evaluation is aimed towards applications taxing high gene detection sensitivity, reproducibility between samples, and minimum hands-on time, as is required, for example, in clinical use. We included two commercial kits, NEBNext® Single Cell/ Low Input RNA Library Prep Kit (NEB®), SMART-seq® HT kit (Takara®), and the non-commercial protocols Genome & Transcriptome sequencing (G&T) and SMART-seq3 (SS3). G&T delivered the highest detection of genes per single cell. SS3 presented the highest gene detection per single cell at the lowest price. Takara® kit presented similar high gene detection per single cell, and high reproducibility between samples, but at the absolute highest price. NEB® delivered a lower detection of genes but remains an alternative to more expensive commercial kits.ConclusionFor the tested kits we found that ease-of-use came at higher prices. Takara can be selected for its ease-of-use to analyse a few samples, but we recommend the cheaper G&T-seq or SS3 for laboratories where a substantial sample flow can be expected.

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Full Length Transcriptome Highlights the Coordination of Plastid Transcript Processing.

Project description:Plastid gene expression involves many post-transcriptional maturation steps resulting in a complex transcriptome composed of multiple isoforms. Although short-read RNA-Seq has considerably improved our understanding of the molecular mechanisms controlling these processes, it is unable to sequence full-length transcripts. This information is crucial, however, when it comes to understanding the interplay between the various steps of plastid gene expression. Here, we describe a protocol to study the plastid transcriptome using nanopore sequencing. In the leaf of Arabidopsis thaliana, with about 1.5 million strand-specific reads mapped to the chloroplast genome, we could recapitulate most of the complexity of the plastid transcriptome (polygenic transcripts, multiple isoforms associated with post-transcriptional processing) using virtual Northern blots. Even if the transcripts longer than about 2500 nucleotides were missing, the study of the co-occurrence of editing and splicing events identified 42 pairs of events that were not occurring independently. This study also highlighted a preferential chronology of maturation events with splicing happening after most sites were edited.

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Benchmarking full-length transcript single cell mRNA sequencing protocols

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2024-01-01 | GSE233170 | GEO

A Unified Nanopore Platform for Direct Sequencing of Individual Full Length RNA Strands

Project description:A Unified Nanopore Platform for Direct Sequencing of Individual Full Length RNA Strands

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A Unified Nanopore Platform for Direct Sequencing of Individual Full Length RNA Strands

Project description:A Unified Nanopore Platform for Direct Sequencing of Individual Full Length RNA Strands

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High-throughput and high-sensitivity full-length single-cell RNA-seq analysis on third-generation sequencing platform

Project description:We developed SCAN-seq2, a high-throughput and highly sensitive single-cell RNA sequencing method based on the TGS platform. Our study demonstrated that SCAN-seq2 improves upon the previous method, SCAN-seq, in terms of sensitivity and throughput. By using reference-guided assembly of single-cell data, we were able to identify thousands of novel full-length RNA isoforms, including cell type-specific expression patterns of pseudogenes. We also accurately determined V(D)J rearrangement events in T and B cells. Lastly, we found that treatment of HepG2 and Hela cells with the spliceosome inhibitor Isoginkgetin (IGG) resulted in a subpopulation of cells with distinct apoptosis features. Our study provides a promising new tool for single-cell transcriptome research. The source code for SCAN-seq2 data analysis pipelines is available at https://github.com/liuzhenyu-yyy/SCAN-seq2 .

2022-11-15 | GSE203561 | GEO

RNA-seq of rat dorsal root ganglia after ligation to investigate central nervous system transcriptomics in chronic pain using agnostic splice site discovery methods

Project description:The study pursued dual goals: To advance mRNA-seq bioinformatics towards unbiased transcriptome capture and to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 dorsal root ganglion (DRG) 2 weeks after L5 spinal Nerve Ligation (SNL). A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJ) achieved a precision of 97%. mRNA-seq of L4 DRG 2 weeks and 2 months after L5 spinal nerve ligation. CONTROL and SNL were used to identify differential gene expression between chronic pain and standard conditions in Rattus norvegicus. CONTROL and SNL and PILOT were used to perform 'agnostic splice site discovery' in the nervous system transcriptome in Rattus norvegicus

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