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Published on: November 14, 2019
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Optimized identification and characterization of small RNAs with PANDORA-seq
Junchao Shi1, Yunfang Zhang2,3, Yun Li4
1China National Center for Bioinformation and Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China. shijc@cncb.ac.cn.
Nature Protocols
|April 3, 2025
Summary
PANDORA sequencing overcomes challenges in small RNA detection by using enzymatic treatments and a specialized pipeline. This method enables comprehensive analysis of diverse small noncoding RNAs (sncRNAs), including those from transfer and ribosomal RNAs.
Area of Science:
- Molecular Biology
- Genomics
- RNA Biology
Background:
- Small noncoding RNAs (sncRNAs) encompass diverse classes like siRNAs, miRNAs, and piRNAs.
- sncRNAs possess varied termini and modifications that impede standard cDNA library construction and sequencing.
- This hinders the detection and comprehensive analysis of many sncRNA types.
Purpose of the Study:
- To introduce PANDORA sequencing, a refined methodology for enhanced sncRNA detection.
- To enable comprehensive analysis of diverse sncRNAs, including those derived from structured RNAs.
- To overcome limitations of standard small RNA sequencing methods.
Main Methods:
- PANDORA sequencing involves sequential enzymatic treatments (T4PNK, AlkB) of size-selected RNAs.
- These treatments address ligation-blocking termini and reverse transcription-blocking RNA modifications.
- Followed by tailored library construction, deep sequencing, and analysis using the SPORTS pipeline.
Main Results:
- PANDORA sequencing effectively circumvents challenges posed by sncRNA termini and modifications.
- The SPORTS pipeline allows comprehensive analysis of various sncRNA types, including those from tRNA and rRNA.
- It also identifies the parental RNA locations of these sncRNAs.
Conclusions:
- PANDORA sequencing offers a transformative tool for exploring the small RNA universe.
- It facilitates a deeper understanding of sncRNA diversity and function.
- This method expands the scope of detectable sncRNAs beyond traditionally studied classes.
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