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Updated: Aug 7, 2025
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Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons
Published on: August 29, 2014
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Sub genomic analysis of SARS-CoV-2 using short read amplicon-based sequencing.
Lian Chye Winston Koh1,2, Yiqi Seow2,3, Kiat Whye Kong3
1Bioinformatics Institute, Agency for Science Technology and Research, Singapore.
Frontiers in Genetics
|March 13, 2023
Summary
This study presents a new sequencing workflow for COVID-19 genetic characterization. The method efficiently identifies subgenomic RNA, aiding in tracking viral load during outbreaks.
Area of Science:
- Virology
- Genomics
- Public Health
Background:
- The COVID-19 pandemic presents a significant global health challenge.
- Accurate and rapid genetic characterization of SARS-CoV-2 is crucial for outbreak management.
- Existing sequencing workflows may not be suitable for decentralized or rapid deployment.
Purpose of the Study:
- To develop and validate a modified sequencing workflow for COVID-19 genetic analysis.
- To demonstrate the workflow's capability in identifying subgenomic RNA characteristics.
- To enable comprehensive genetic characterization during public health emergencies.
Main Methods:
- Utilized a short tiling amplicon library preparation method (280bp).
- Paired the method with Illumina's iSeq100 desktop sequencer.
- Developed Taqman assays based on sequencing data to quantify subgenomic RNA.
Main Results:
- Successfully identified gapped reads indicative of subgenomic RNA junctions in patient samples.
- The workflow proved versatile, small-footprint, and suitable for decentralized use.
- Taqman assays accurately quantified subgenomic ORF5 and ORF7a RNA.
- Combined approach effectively tracked subgenomic RNA titres alongside standard qRT-PCR.
Conclusions:
- The modified sequencing workflow offers a versatile solution for decentralized COVID-19 genetic characterization.
- The ability to identify and track subgenomic RNA is valuable for understanding viral dynamics.
- This approach enhances capabilities for comprehensive genetic analysis during infectious disease outbreaks.
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