Amplicon-based sequencing is a powerful next-generation sequencing (NGS) technique utilised to detect and identify bacteria and archaea within biological samples. This method is particularly effective for screening genetic variants and target microorganisms, enabling comprehensive analysis and comparison of biodiversity across a wide range of complex environments. It is extensively applied in microbial ecology for studying populations and communities, reconstructing the phylogeny of specific microbial groups, identifying species in mixed cultures, and detecting key organisms, whether pathogenic or beneficial.
Targeting Ribosomal RNA Regions for Microbial Identification
This approach utilises conserved ribosomal RNA regions to design primers targeting variable regions between them. The resulting PCR products, known as amplicons, are then sequenced. These primers target the variable regions (V1-9) between the conserved sequences, which are often genus- or species-specific. As a result, microorganisms can be accurately identified at the genus level, with some resolution down to the species level. For bacterial and archaeal identification, 16S amplicon sequencing is commonly used, focusing on the 16S rRNA gene, a highly conserved marker across these domains.
Increasing Resolution with Hypervariable Region Combinations
Combinations of the 9 hypervariable regions within the 16S rRNA gene are often sequenced together to increase the resolving power of the 16S rRNA sequencing method. For example, the Quick-16S™ Plus NGS library prep kit from ZymoBIOMICS has options for sequencing the V1-V2, V1-V3, V3-V4 and V4 regions. Sequencing more than one region at a time increases the resolution and provides increased certainty of taxonomic composition of your sample.
Advantages of the Quick-16S™ Plus NGS Library Prep Kit
The Quick-16S™ Plus NGS library prep kit stands out as the fastest and easiest 16S library prep solution, allowing you to convert up to 96 DNA samples into a sequence-ready 16S rRNA library with just 30 minutes of hands-on time. By incorporating real-time, quantitative PCR (qPCR), this kit facilitates direct quantification of PCR products, removing the need for extra library quantification methods like gel electrophoresis. After equal volume pooling, a single clean-up step completes the final library preparation.
Outsourced 16S rRNA Sequencing Services by ZymoBIOMICS
16S rRNA sequencing is also available as an outsourced service from ZymoBIOMICS, with their complete workflow covering everything from nucleic acid extraction, library preparation, sequencing and data analysis. By combining leading edge sequencing error correction methods with a highly curated 16S rRNA database, ZymoBIOMICS’ 16S amplicon sequencing service can provide taxonomic identification down to the species level.
If you are interested in preparing your own libraries or outsourcing your 16S amplicon sequencing, you can contact our genomics specialists to discuss your project.