Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

Alternative RNA Splicing

Alternative RNA Splicing

Alternative RNA splicing is the regulated splicing of exons and introns to produce different mature mRNAs from a single pre-mRNA. Unlike in constitutive splicing where a single gene produces a single type of mRNA, alternative splicing allows an organism to produce multiple proteins from a single gene and plays an important role in protein diversity.
There are five types of alternative RNA splicing that vary in the ways the pre-mRNA segments are removed or retained in the mature mRNA. The first...

Alternative RNA Splicing

Alternative RNA Splicing

Pre-mRNA Processing: Modification of pre-mRNA Ends

Pre-mRNA Processing: Modification of pre-mRNA Ends

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a cap to the 5' end of the growing transcript. In this process, a 5' phosphate is replaced by modified guanosine that has a methyl group attached (7-methyl guanosine). This 5' cap helps...

RNA Splicing

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...

pre-mRNA Processing

pre-mRNA Processing

In eukaryotic cells, transcripts made by RNA polymerase are modified and processed before exiting the nucleus. Unprocessed RNA is called precursor mRNA or pre-mRNA to distinguish it from mature mRNA.
Once about 20-40 ribonucleotides have been joined together by RNA polymerase, a group of enzymes adds a “cap” to the 5’ end of the growing transcript. In this process, a 5’ phosphate is replaced by modified guanosine that has a methyl group attached to it (7-Methyl...

Eukaryotic RNA Polymerases

Eukaryotic RNA Polymerases

RNA Polymerase (RNAP) is conserved in all animals, with bacterial, archaeal, and eukaryotic RNAPs sharing significant sequence, structural, and functional similarities. Among the three eukaryotic RNAPs, RNA Polymerase II is most similar to bacterial RNAP in terms of both structural organization and folding topologies of the enzyme subunits. However, these similarities are not reflected in their mechanism of action.
All three eukaryotic RNAPs require specific transcription factors, of which the...

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Canine Fecal Microbiome Dataset: Ultra-deep Multi-platform Sequencing Across Extraction and Library Protocols.

Scientific data·2026

Same author

Multi-platform profiling reveals host- and cell -type-specific pseudorabies virus gene expression.

Scientific reports·2026

Same author

Veterinary Herpesviruses: Experimental Tools for Transcriptomics and Neuroscience.

Veterinary sciences·2026

Same author

Effect of long-lasting antimicrobial surface sprays in real-life environment.

BMC infectious diseases·2026

Same author

Longitudinal long-read microbiome profiling in a canine model reveals how age, diet, and birth mode shape gut community dynamics.

mSystems·2026

Same author

Genome sequence of a human monkeypox virus isolate from Central Europe during the 2022 outbreak.

Microbiology resource announcements·2025

Search research articles

Related Experiment Video

Updated: Jan 4, 2026

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

Published on: June 24, 2021

Template-switching artifacts resemble alternative polyadenylation.

Zsolt Balázs¹, Dóra Tombácz^1,2, Zsolt Csabai¹

¹Department of Medical Biology, Faculty of Medicine, University of Szeged, Szeged, Hungary.

|November 10, 2019

Summary

Template-switching artifacts in cDNA sequencing can create spurious alternative polyadenylation (APA) sites. A new algorithm effectively filters these artifacts, improving APA analysis accuracy, especially when compared to direct RNA sequencing.

Keywords:

Direct RNA sequencing Internal priming Long-read sequencing Polyadenylation RNA sequencing Template switching cDNA sequencing

More Related Videos

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Published on: April 26, 2017

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Published on: October 9, 2014

Related Experiment Videos

Last Updated: Jan 4, 2026

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

Published on: June 24, 2021

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Published on: April 26, 2017

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Published on: October 9, 2014

Area of Science:

Molecular Biology
Genomics
Bioinformatics

Background:

Alternative polyadenylation (APA) is frequently studied using cDNA sequencing.
cDNA sequencing is susceptible to template-switching artifacts.
These artifacts are often overlooked, with APA errors misattributed to internal priming.

Purpose of the Study:

To investigate the impact of template-switching artifacts on APA analysis.
To develop and validate a novel algorithm for filtering spurious polyadenylation sites in cDNA sequencing data.
To compare the performance of the new algorithm against conventional internal priming filters.

Main Methods:

Analysis of long-read cDNA and direct RNA sequencing data from two organisms across different platforms.
Development of a filtering algorithm accounting for template-switching artifacts.
Comparative analysis of the new algorithm with existing internal priming filters using direct RNA sequencing data as a benchmark.

Main Results:

The developed filtering algorithm successfully identified and removed artifactual polyadenylation sites caused by template switching.
The algorithm demonstrated superior performance compared to conventional internal priming filters.
Artifactual polyadenylation in cDNA sequencing was observed even with short runs of adenines (≥3).
No significant difference in poly(A) tail length was found between artifactual and true polyadenylation sites, contrary to expectations for internal priming artifacts.

Conclusions:

Template switching is a significant contributor to spurious polyadenylation site generation in cDNA sequencing.
There is a critical need for improved filtering of artifactual polyadenylation sites in cDNA datasets.
Direct RNA sequencing offers a valuable alternative for accurate APA annotation, bypassing cDNA-related artifacts.