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Related Experiment Video

Updated: May 7, 2026

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

Published on: August 29, 2014

Organism-specific rRNA capture system for application in next-generation sequencing.

Sai-Kam Li¹, Jun-Wei Zhou, Aldrin Kay-Yuen Yim

¹Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR.

|September 28, 2013

Summary

This study introduces a computer-designed, organism-specific probe method for efficient ribosomal RNA (rRNA) depletion in RNA sequencing. This approach improves RNA integrity and abundance, crucial for studying low-expression genes in microbes.

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Area of Science:

Molecular Biology
Bioinformatics
Genomics

Background:

RNA sequencing (RNA-seq) is vital for RNomics but hindered by high ribosomal RNA (rRNA) and transfer RNA (tRNA) abundance.
Lowly expressed genes are difficult to study due to rRNA/tRNA predominance, necessitating rRNA depletion before sequencing.
Current rRNA depletion methods using hybridization probes can cause non-specific binding and unintended messenger RNA (mRNA) removal, varying by organism.

Purpose of the Study:

To develop a computational method for designing organism-specific probes to deplete rRNA.
To evaluate the efficacy of these custom probes for rRNA removal in RNA sequencing.
To compare the performance of organism-specific probes against commercial kits for RNA integrity and abundance preservation.

Main Methods:

A computer-based algorithm was developed to design organism-specific RNA probes for rRNA depletion.
Biotinylated RNA probes were synthesized via in vitro transcription based on computational design.
Subtractive hybridization was employed using the designed probes for rRNA removal, exemplified in Mycobacterium smegmatis.

Main Results:

The developed organism-specific probes efficiently removed 16S and 23S rRNAs from Mycobacterium smegmatis.
Compared to a commercial kit, the organism-specific probe method demonstrated superior preservation of RNA integrity and abundance.
The computational design approach proved effective for targeted rRNA depletion.

Conclusions:

A computer-based design strategy enables the creation of effective, organism-specific rRNA-depleting probes.
This method offers an improvement over commercial kits by minimizing unintended mRNA loss and preserving RNA quality.
The approach is proposed as a generic solution for preparing RNA samples for next-generation sequencing, especially for microbial transcriptome analysis.