What is Degradome Sequencing (Degradome-Seq)?

Degradome Sequencing, also known as Parallel Analysis of RNA Ends (PARE), is a high-throughput method used to study RNA degradation products, especially those cleaved by microRNAs (miRNAs) or small interfering RNAs (siRNAs). It provides a snapshot of RNA decay events, revealing how gene expression is regulated post-transcriptionally.

Degradome-Seq allows researchers to map the precise cleavage sites on messenger RNAs and discover target genes regulated by small RNAs.

Overview

• Purpose: To identify and analyze RNA cleavage events

• Focus: mRNA degradation products (5′ uncapped RNA ends)

• Main Use: Confirming miRNA or siRNA cleavage targets

• Technology Used: Next-Generation Sequencing (NGS)

How Degradome Sequencing Works

1. Total RNA Extraction
   Total RNA is isolated from cells or tissues.

2. Enrichment of 5′-monophosphorylated RNA
   Captures uncapped RNA degradation products (typically created after cleavage).

3. Adapter Ligation & Reverse Transcription
   Specific adapters are ligated to the 5′ ends of degraded RNAs, followed by cDNA synthesis.

4. PCR Amplification & Library Construction
   The cDNA is amplified and prepared into a library suitable for NGS.

5. Sequencing
   The library is sequenced, usually with single-end reads.

6. Bioinformatics Analysis
   Reads are aligned to the reference transcriptome to identify cleavage sites, often corresponding to miRNA target locations.

Applications of Degradome-Seq

• miRNA Target Validation
  Confirms direct cleavage of mRNAs by specific microRNAs.

• Post-transcriptional Regulation Studies
  Identifies degradation patterns and decay intermediates.

• Plant Gene Silencing Research
  Widely used in plant biology where miRNA cleavage is a common regulatory mechanism.

• Transcript Stability Profiling
  Helps measure the half-lives of different mRNA transcripts.

• Functional Annotation of Non-coding RNAs
  Assesses how small RNAs affect gene regulation at the transcript level.

Key Features of Degradome-Seq

Advantages of Degradome Sequencing

• High specificity for 5′-cleaved RNA ends

• Enables direct identification of miRNA targets

• No prior knowledge of targets required

• High-throughput, genome-wide coverage

• Applicable to a wide range of species

Limitations and Challenges

• Requires High-Quality RNA

• Lower sensitivity for targets cleaved at low frequency

• Not all RNA decay intermediates are stable enough to be captured

• Complex data interpretation — needs custom tools and databases

• Works best when paired with miRNA-Seq data

Tools & Software for Degradome Data Analysis

• Cleaveland – A popular tool for mapping degradome data to predicted miRNA targets

• PAREsnip – High-throughput degradome data analysis, especially for plant miRNAs

• sPARTA – Advanced tool for miRNA target prediction and degradome validation

• TargetFinder – Predicts miRNA targets; useful when paired with degradome data

• PsRobot – Small RNA target prediction in plants using degradome evidence

Who Uses Degradome-Seq?

• Plant Biologists – To validate miRNA-mRNA cleavage in Arabidopsis, rice, maize, etc.

• Genomics Researchers – Investigating transcript degradation and stability

• Bioinformaticians – Integrating degradome data with transcriptomic datasets

• Epigeneticists – Exploring RNA silencing mechanisms

• Pharmaceutical Researchers – Studying RNA turnover in drug response or toxicity

Comparison: Degradome-Seq vs Other RNA Techniques