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Updated: Jun 13, 2025

3' End Sequencing Library Preparation with A-seq2

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Published on: October 10, 2017

StringTie3 Improves Total RNA-seq Assembly by Resolving Nascent and Mature Transcripts.

Ida Shinder^1,2, Geo Pertea³, Richard Hu^2,4

¹Cross Disciplinary Graduate Program in Biomedical Sciences, Johns Hopkins School of Medicine, Baltimore, MD, United States.

Biorxiv : the Preprint Server for Biology

|June 12, 2025

Summary

StringTie3 accurately assembles total RNA sequencing (RNA-seq) data by distinguishing nascent from mature transcripts. This new method improves RNA-seq assembly accuracy and reveals novel insights into gene regulation.

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Area of Science:

Genomics
Molecular Biology
Bioinformatics

Background:

Accurate transcriptome assembly from total RNA sequencing (RNA-seq) is hindered by the conflation of nascent and mature RNA isoforms.
Existing methods lead to misassemblies and quantification errors, impacting downstream biological analyses.

Purpose of the Study:

To introduce StringTie3, a novel assembler for total RNA-seq data that addresses the challenge of distinguishing nascent from mature transcripts.
To improve the accuracy and sensitivity of RNA-seq assembly, particularly for long-read and hybrid datasets.

Main Methods:

StringTie3 incorporates a "nascent mode" to model co-transcriptional splicing and separate nascent from mature transcripts.
A refined long-read module differentiates genuine polyadenylation sites from artifacts.
Performance was evaluated across short-, long-, and hybrid-read datasets using various biological experiments.

Main Results:

StringTie3 significantly reduces assembly errors compared to existing tools.
It improves short-read total RNA-seq precision by up to 20% and enhances long-read assembly sensitivity and precision by up to 37% and 75%, respectively.
Analysis of Argonaute knockouts and breast cancer samples revealed distinct patterns of nascent and mature RNA regulation.

Conclusions:

StringTie3 provides a powerful framework for accurate transcriptome reconstruction from total RNA-seq data.
The ability to distinguish nascent from mature RNA uncovers previously hidden layers of transcriptional and post-transcriptional regulation.
This tool enhances the investigation of complex RNA processing and regulatory mechanisms.