The culture-free study of microbial community composition has been revolutionized by the application of high-throughput, low-cost, molecular techniques. The major approaches used for taxonomic studies and the abundance of complex microbiomes or environments are ribosomal RNA and shotgun sequencing. Results obtained from both methods are useful for analyzing the taxonomic composition of microbial communities. However, they differ in their recovery efficiency, number of required reads to obtain accurate taxonomic profiles, ability to resolve at the genus and species level, and usefulness to study functional genes. Therefore, to choose one that suits your study goals, it is essential to consider the pros and cons of Amplicon-based rRNA (mainly 16S rRNA) vs Shotgun Metagenomics.

In this blog, we’ll focus on comprehensive understanding of both methods, and which one we should choose to achieve our research goals.

16S/18S/ITS Amplicon Metagenomic Sequencing 

16S/18S/ITS Amplicon Metagenomic Sequencing is a DNA sequencing method that targets specific regions (amplicons) for in-depth analysis. This approach involves amplifying short (<500 bp) hypervariable regions of conserved genes or intergenic regions using PCR, followed by next-generation sequencing (NGS). This method enables the identification and differentiation of multiple microbial species within complex samples. Amplicon metagenomic sequencing specifically targets the 16S ribosomal RNA (rRNA) gene for bacteria and archaea, the 18S rRNA gene for eukaryotes, and the Internal Transcribed Spacer (ITS) regions for fungi (including yeasts, molds, and other related species). This allows for comprehensive phylogenetic and taxonomic analysis.

Figure 1: Marker genes for Amplicon Metagenomic Sequencing

Shotgun Metagenomic Sequencing 

Shotgun Metagenomic Sequencing refers to the use of random, “shotgun” sequencing of microbial DNA. This approach can resolve at the species and subspecies level. It has advantages over rRNA analysis of detecting viruses, bacteria, archaea, and eukaryotes, does not require primer selection, and can provide information about entire genomes. When using this approach, Novogene can assist researchers in their discovery efforts by analyzing cellular processes, environmental information, human diseases, metabolism, organismal systems, and antibiotic gene annotation.

Factors for consideration: Amplicon vs Shotgun sequencing

1. Sample type

Host DNA interference of samples is a critical factor in choosing a sequencing method. Since Amplicon Metagenomic Sequencing uses targeted primers to amplify only DNA of interest, there is a lower risk of host DNA being unintentionally amplified. In contrast, Shotgun Metagenomic Sequencing captures all DNA present in a sample, so non-microbial reads might dominate and overlook microbiome data.

Although host DNA depletion (for example, by differential lysis of human and bacterial cells) can be performed before the library preparation for Shotgun Sequencing, this approach may leave insufficient microbial genomic DNA after depletion. Therefore, for samples with a high ratio of host to microbial DNA like skin, tissue or blood, 16S rRNA gene sequencing is often more appropriate. In high-biomass samples like feces or soil, either method can work.

Figure 2: Samples with low vs high host DNA

2. Taxonomic resolution and coverage

The choice between 16S rRNA and Shotgun Metagenomic Sequencing often comes down to what you’re trying to achieve – whether you need broad microbial coverage, fine taxonomic resolution, or a balance of both.

In the case of 16S rRNA sequencing, taxonomic resolution is typically limited to the genus level (e.g., Bifidobacteria) when using short-read sequencing platforms. Long-read sequencing can improve resolution to the species level, but the accuracy still depends on several factors: the specific region of the 16S gene targeted (e.g. V3-V4 or V5-V6), the diversity of the organism being analyzed, and the bioinformatics pipeline used for sequence analysis.

By contrast, Shotgun Metagenomic Sequencing provides a much more comprehensive view. This method allows for the detection of a wider range of organisms—including bacteria, archaea, fungi, protists, viruses, and even host DNA—without the amplification bias introduced by primer selection. It also offers higher taxonomic resolution, often identifying microbes down to the species level (e.g., Bifidobacterium longum) and, in many cases, the strain level (e.g., Bifidobacterium longum 35624). This is made possible through genome-wide profiling and the analysis of single nucleotide variants (SNVs).

While Shotgun Metagenomics provides a more complete picture of microbial communities, Amplicon Sequencing remains a practical and effective tool for focused studies – particularly when the goal is to characterize specific microbial groups at moderate taxonomic resolution.

3. Costs

Although Shotgun Metagenomic Sequencing provides much more data than 16S rRNA gene sequencing, you will have to pay for that extra data. To work around this data vs cost dilemma, some researchers perform 16S rRNA gene sequencing on all their samples in addition to Shotgun Metagenomic Sequencing on a small subset of samples in their study. The cost per sample of sequencing also depends on sequencing depth. 

4. Composition vs function

Microbiome research is increasingly shifting beyond simply cataloging microbial taxa (taxonomic composition) to understanding the functional potential of microbial communities. Large-scale human microbiome studies have shown that functional metagenomic data, which reveal the genes and pathways present, can offer greater power to distinguish between ‘healthy’ and ‘diseased’ microbiomes than taxonomy alone. While 16S rRNA gene sequencing does not directly measure microbial genes, some bioinformatics tools such as PICRUSt that is included in Novogene’s QIIME 2 analysis pipeline, can be used to infer functional profiles from 16S data. However, these predictions are inherently limited and lack the resolution of direct gene-level data.

For researchers interested in actual microbial gene content, such as antibiotic resistance genes or virulence factors, Shotgun Metagenomic Sequencing is the more appropriate approach. Novogene offers two types of shotgun metagenomics services:

• Deep Shotgun Metagenomics (Assembly-Based): This method reconstructs microbial genomes and is ideal for in-depth functional profiling, including detailed analysis of antibiotic resistance genes and novel gene prediction.
• Shotgun Metagenomics Reads-Mapping: This faster, reference-based approach maps sequencing reads to known databases in case researchers only want to look into function annotation across known genes.

Both approaches enable comprehensive insights into microbial function, but the assembly-based method offers more robust capabilities for gene discovery and resistance profiling.

Figure 3: Novogene’s demo analysis result for Shotgun sequencing.

5. Summary

Factors	16S/18S/ITS rRNA sequencing	Shotgun Metagenomics (Assembly-Based)	Shotgun Metagenomics (Reads-Mapping)
Host DNA Contamination	Low (but PCR success depends on the absence of inhibitors and the presence of a detectable microbiome)	High, varies with sample type (but this can be mitigated by calibrating the sequencing depth)	High, varies with sample type (but this can be mitigated by calibrating the sequencing depth)
Functional Profiling (profile microbial genes)	No (but ‘predicted’ functional profiling is possible)	Yes (but it only reveals information on functional potential)	Yes (but it only reveals information on functional potential)
Cost	More cost-effective	2-3x	2-3x
Taxonomic Resolution	Genus (sometimes species/ sub-species if full-length-sequencing); dependent on region(s) targeted	Species (sometimes strains and single nucleotide variants, if sequencing is deep enough)	Species (sometimes strains and single nucleotide variants, if sequencing is deep enough)
Taxonomic Coverage	Amplicon-specific: + 16S rRNA: Bacteria and archaea + 18S rRNA: Protist and algae + ITS: Fungi (including yeasts, molds, and other related species)	All taxa, including viruses	All taxa, including viruses
Taxonomy and Function Annotation	✓	✓	✓
Antibiotic Resistance Annotation	x	✓	x

6. Novogene services

Novogene offers comprehensive, end-to-end sequencing services tailored to the specific needs of a study. Their services include sample preparation, sequencing, bioinformatics analysis, and interpretation, ensuring researchers receive detailed insights into microbial community composition and function.

As an official distributor of Novogene in Vietnam, GeneSmart is committed to making advanced genomic sequencing services and expert support from Novogene more accessible to the Vietnamese scientific community.

Stay tuned for our next blog, where we’ll dive deeper into bioinformatics pipelines and explore practical applications of both 16S/18S/ITS Amplicon and Shotgun Metagenomic Sequencing.

Learn more about Novogene’s 16S/18S/ITS Amplicon and Shotgun Metagenomic Sequencing services at GeneSmart here

References

1. Novogene. 16S rRNA and Shotgun: Approaches to Metagenomics. Available at: https://www.novogene.com/amea-en/resources/blog/16s-rrna-and-shotgun-approaches-to-metagenomics/
2. Gupta, A. Metagenomics vs. 16S sequencing: Decoding microbial communities. Available at: https://www.linkedin.com/pulse/metagenomics-vs-16s-sequencing-decoding-microbial-communities-j74ue/
3. Microbiome Insights. 16S rRNA sequencing vs. shotgun metagenomic sequencing. Available at: https://blog.microbiomeinsights.com/16s-rrna-sequencing-vs-shotgun-metagenomic-sequencing#taxonomic-resolution

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