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RNA-seq

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases.
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...

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Related Experiment Video

Updated: Sep 24, 2025

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Published on: May 21, 2020

UNAGI: Yeast Transcriptome Reconstruction and Gene Discovery Using Nanopore Sequencing.

Mohamad Al Kadi¹, Nicolas Jung², Daisuke Okuzaki^3,4,5

¹Department of Bacterial Infections, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.

Methods in Molecular Biology (Clifton, N.J.)

|May 6, 2022

Summary

Long-read sequencing, like Oxford Nanopore, enhances genome annotation accuracy by directly sequencing full-length complementary DNA (cDNA). This method improves transcriptome reconstruction and identifies novel noncoding RNAs, overcoming limitations of traditional short-read sequencing.

Keywords:

Full-length cDNA Long-read sequencing Nanopore Pipeline Transcriptome reconstruction

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Last Updated: Sep 24, 2025

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

Published on: May 21, 2020

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Published on: June 3, 2019

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Published on: March 11, 2020

Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Traditional computational genome annotation methods suffer from low accuracy, failing to identify untranslated regions, predict complex isoforms, and discover noncoding RNAs.
RNA sequencing (RNA-seq) improved transcriptome reconstruction but cDNA fragmentation in short-read sequencing leads to information loss and assembly inaccuracies.
Long-read sequencing technologies, such as Oxford Nanopore, offer direct cDNA sequencing without fragmentation, preserving transcript structure and enhancing reconstruction accuracy.

Purpose of the Study:

To present a protocol and bioinformatics pipeline for accurate transcriptome reconstruction of compact genomes, including yeasts.
To leverage long-read sequencing for improved identification of transcripts, isoforms, and novel RNAs.
To enhance the accuracy of existing genome annotations.

Main Methods:

Generation of full-length complementary DNA (cDNA).
Utilizing Oxford Nanopore ligation-based sequencing for multiplexed sample runs.
A bioinformatics pipeline involving read stranding, reference-guided correction, transcript identification (including 5' and 3' UTRs), isoform profiling, and annotation refinement.

Main Results:

The protocol and pipeline successfully reconstruct transcriptomes from compact genomes.
Long-read sequencing significantly improves transcriptome reconstruction accuracy compared to traditional methods.
A substantial number of novel RNAs were discovered, and annotation accuracy was enhanced.

Conclusions:

Long-read sequencing directly addresses limitations of short-read RNA-seq for accurate transcriptome reconstruction.
The presented protocol and pipeline provide an effective solution for annotating compact genomes.
This approach enhances the discovery of novel transcripts and improves the overall quality of genome annotations.