Exploring Microbiome Sequencing Techniques

As the field of biotechnology progresses into its next decade, the understanding of the microscopic world and how it interacts with the surrounding macroscopic environment continues to develop rapidly. The rise of next-generation sequencing (NGS) technologies has made genetic sequencing more robust, affordable, and accessible. This increased accessibility opens new avenues for researchers to explore microbiome sequencing and understand complex relationships within microbial communities. It also fuels the expanding field of bioinformatics, as terabytes of sequencing data are continually generated and analysed.

Two of the most notable short-read NGS techniques for producing high-quality, comparable, and reproducible data are targeted 16S rRNA gene sequencing and shotgun metagenomic sequencing. While each workflow has key distinguishing features, there is also overlap in the type of data generated. Understanding these similarities and differences is essential for researchers using NGS microbiome analysis, as these factors will influence both cost and capability.

This article compares these two major microbiome sequencing techniques, considering factors such as taxonomic resolution, depth and breadth of profiling, cost, and workflow robustness. The aim is to provide scientists with clear, practical information to guide their choice between targeted 16S rRNA sequencing and shotgun metagenomic sequencing.

Shotgun metagenomic sequencing, often referred to as whole genome sequencing (WGS), involves indiscriminate sequencing of all genetic material present in a sample. This may include DNA from all domains of life - viruses, archaea, prokaryotes, and eukaryotes - as well as host genetic material if no selection step is applied. Although library preparation methods vary, the fundamental process involves shearing input DNA into fragments of appropriate size, ligating sequencing adapters, and adding barcode index sequences via PCR. The final products are cleaned, pooled, and ready for sequencing.

A typical workflow for taxonomic analysis of shotgun metagenomic data includes trimming to remove adapter sequences and low-quality reads, filtering to exclude host, contaminant, and low-complexity reads, and comparing against reference databases (e.g. Kraken2, Centrifuge, MetaPhlAn, mOTUs). This generates a taxonomic profile covering all organisms present in a microbiome, enabling comprehensive cataloguing. In addition to taxonomic identification, shotgun sequencing can reveal functional pathways and detect genes associated with antibiotic resistance.

Both shotgun metagenomic sequencing and targeted 16S sequencing are widely used for microbiome profiling. Since microbes inhabit virtually every environment, potential applications range from environmental studies to human microbiome research. Depending on the research goal, scientists may want to identify the organisms present, assess diversity and abundance, or investigate potential metabolic activity.

Targeted 16S sequencing is limited to the amplified gene regions, making it suitable for focused investigations or for parallel sequencing targeting other regions (e.g. ITS for fungi). Shotgun sequencing, in contrast, covers all genetic material, enabling detection of organisms without the targeted gene and offering higher taxonomic resolution, sometimes down to strain level. It also provides functional potential insights, although confirming activity requires further techniques such as metatranscriptomics.

Operational demands: WGS requires substantial investment in sequencing instruments, reagents, and data storage. The large datasets generated often necessitate advanced bioinformatics infrastructure. Targeted 16S sequencing produces smaller datasets and is less expensive to run, making it accessible for smaller labs or those outsourcing sequencing to providers.

Selecting the right microbiome sequencing method is essential for successful microbiome research. Targeted 16S rRNA sequencing is well suited to initial microbial profiling, particularly when dealing with low-input or host-rich samples. Shotgun metagenomic sequencing offers unparalleled resolution and functional potential insights but comes with higher costs and technical demands.

For many laboratories, outsourcing microbiome sequencing to experienced providers offers a cost-efficient way to access high-quality data without major capital investment. Providers such as Zymo Research offer end-to-end solutions from sample processing to bioinformatics analysis.

As sequencing technologies evolve, researchers can expect continued improvements in accuracy, cost, and accessibility, broadening the scope of microbiome research worldwide.

This article is based on an original post by Zymo Research. You can read the original here.

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