Dataset Information

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

ABSTRACT: RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

SUBMITTER: Begik O

PROVIDER: S-EPMC9834059 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

Begik Oguzhan O Diensthuber Gregor G Liu Huanle H Delgado-Tejedor Anna A Kontur Cassandra C Niazi Adnan Muhammad AM Valen Eivind E Giraldez Antonio J AJ Beaudoin Jean-Denis JD Mattick John S JS Novoa Eva Maria EM

Nature methods 20221219 1

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq ...[more]

PMID: 36536091

Similar Datasets

Project description:Transcriptome profiling is a mainstay of translational cancer research and is increasingly finding its way into precision oncology. While bulk RNA sequencing (RNA-seq) is widely available, high investment costs and long data return time are limiting factors for clinical applications. We investigated a portable nanopore long-read sequencing device (MinION, Oxford Nanopore Technologies) for transcriptome profiling of tumors. In particular, we investigated the impact of lower coverage than that of larger sequencing devices by comparing shallow nanopore RNA-seq data with short-read RNA-seq data generated using reversible dye terminator technology (Illumina) for ten samples representing four cancer types. Coupled with ShaNTi (Shallow Nanopore sequencing for Transcriptomics), a newly developed data processing pipeline, a turnaround time of five days was achieved. The correlation of normalized gene-level counts between nanopore and Illumina RNA-seq was high for MinION but not for very low-throughput Flongle flow cells (r = 0.89 and r = 0.24, respectively). A cost-saving approach based on multiplexing of four samples per MinION flow cell maintained a high correlation with Illumina data (r = 0.56-0.86). In addition, we compared the utility of nanopore and Illumina RNA-seq data for analysis tools commonly applied in translational oncology: (1) Shallow nanopore and Illumina RNA-seq were equally useful for inferring signaling pathway activities with PROGENy. (2) Highly expressed genes encoding kinases targeted by clinically approved small-molecule inhibitors were reliably identified by shallow nanopore RNA-seq. (3) In tumor microenvironment composition analysis, quanTIseq performed better than CIBERSORT, likely due to higher average expression of the gene set used for deconvolution. (4) Shallow nanopore RNA-seq was successfully applied to detect fusion genes using the JAFFAL pipeline. These findings suggest that shallow nanopore RNA-seq enables rapid and biologically meaningful transcriptome profiling of tumors, and warrants further exploration in precision cancer medicine studies.

Project description:BackgroundViral relapse is a major concern in hepatitis C virus (HCV) antiviral therapy. Currently, there are no satisfactory methods to predict viral relapse, especially in the era of direct acting antivirals in which the virus often quickly becomes undetectable using PCR-based approaches that focus on a small viral region. Next-generation sequencing (NGS) provides an alternative option for viral detection in a genome-wide manner. However, owing to the overwhelming dominance of human genetic content in clinical specimens, direct detection of HCV by NGS has a low sensitivity and hence viral enrichment is required.MethodsBased on template-dependent multiple displacement amplification (tdMDA), an improved method for whole genome amplification (Wang et al., 2017. Biotechniques 63, 21-27), we evaluated two strategies to enhance the sensitivity of NGS-based HCV detection: duplex-specific nuclease (DSN)-mediated depletion of human sequences and HCV probe-based capture sequencing.ResultsIn DSN-mediated depletion, human sequences were significantly reduced in the two HCV serum samples tested, 65.3% → 55.6% → 33.7% (#4727) and 68.6% → 56% → 21% (#4970), respectively for no normalization, self- and driver-applied normalization. However, this approach was associated with a loss of HCV sequences perhaps due to its micro-homology with the human genome. In capture sequencing, HCV-mapped sequencing reads occupied 96.8% (#4727) and 22.14% (#4970) in NGS data, equivalent to 1936x and 7380x enrichment, respectively. Capture sequencing was then applied to ten serum samples collected at the end of HCV antiviral therapy. Interestingly, the number of HCV-mapped reads was significantly higher in relapsed patients (n = 5) than those from patients with sustained virological response (SVR) (n = 5), 102.4 ± 72.3 vs. 2.6 ± 0.55, p = 0.014.ConclusionsOur data provides concept evidence for a highly sensitive HCV detection by capture sequencing. The abundance difference of HCV sequencing reads at the end of HCV antiviral therapy could be applied to predict treatment outcomes.

Dataset Information

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

Publications

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets