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16S rRNA and Shotgun: Approaches to Metagenomics

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 of these 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. There have been noteworthy advances in the techniques, methods of analysis, and their applications. We’ll focus on their respective benefits, and then further our understanding of their application in a study about oral microbiome and SARS-CoV-2 infection.

16S rRNA/18S rRNA/ITS Sequencing

16S rRNA gene sequencing elucidates bacterial archaeal microbiomes based on differences in 16S rRNA gene hypervariable regions. This form of amplicon-based metataxonomic analysis targets areas that are highly conserved among taxa. The 16S rRNA gene is about 1,500 bp long and contains nine hypervariable regions that are typically used for identification at the genus, and sometimes species, level. Park et al found that the use of V1-V2 (27F-337R) and V3 (337F-518R) regions produced the most representative results from a pooled sample of eight different species of bacteria. [1] However, the V3-V4 region may contain the highest level of variability and the target bacterial community should be considered when determining primer selection.

18S rRNA gene sequencing is often targeted for eukaryotic microbiome analysis. This approach is challenging due to similarities between animal hosts and fungal genes. There are a variety of approaches to minimize host contamination including optimized primer design or the use of alternative targets such as the ITS1 segment of the internal transcribed spacer (ITS) region. Thus, the ITS1 region of the ITS is often used to identify fungal species infecting animals and determine the microbiome. Here again, it is best to consider the target microbial community for primer selection. Compared to shotgun analysis these methods of targeting a single, hypervariable gene are useful for smaller samples. However, whereas they are cost-effective and useful they can introduce amplification bias and provide far less information than shotgun metagenomic sequencing.

Shotgun Metagenomic Sequencing

Shotgun metagenomics refers to the use of random, “shotgun” sequencing of microbial DNA. This whole genome sequencing approach can resolve at the species and subspecies level. It has the 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.

Application Note

Many studies have explored the link between the clinical outcomes of SARS-CoV-2 infection and the human microbiome. However, our understanding of changes in the salivary microbiome during different stages of SARS-CoV-2 infection remains limited. In July 2023, researchers from Rutgers University published a pioneering community-based study titled “Saliva microbiome in relation to SARS-CoV-2 infection in a prospective cohort of healthy US adults” in the EBioMedicine journal. This study broadens our understanding of how SARS-CoV-2 infection impacts the salivary microbiome. [2]

Blaser and colleagues examined the variation in upper respiratory tract microbiome using saliva samples from individuals at different stages of SARS-CoV-2 infection. They employed 16S rRNA gene and shotgun metagenomic sequencing methods to conduct a comprehensive analysis of the total bacterial abundance, composition, population structure, and gene function of the microbiome from 748 serial saliva samples. [2] Specifically, they used amplicon sequence variants (ASVs) generated from 16S rRNA gene sequencing to measure taxonomic composition, while functional gene pathways of the microbiome were assessed using shotgun metagenomic sequencing. Novogene provided the DNA library preparations and shotgun metagenomic sequencing services.

Their findings revealed a relative stability in the salivary microbiome in cases of mild to moderate SARS-CoV-2 infections. However, severe infections led to significant reductions in microbiome diversity shortly after infection, indicating the limits of microbiome resilience when faced with severe infection. [2]


Both ribosomal and shotgun approaches to metagenomics have advanced and will continue to advance our knowledge of microbial communities, their roles in pathology, and their important contributions to sustaining health and well-being. The choice of which to use rests largely on sample size and the desired depth of analysis. Besides, meta-transcriptome analysis provides the tool needed to study host-microbiome interactions, and associated mechanisms of disease, and develop treatments and strategies to restore and maintain health. Future discoveries will likely be facilitated by in-depth analysis afforded by functional metagenomics and meta-transcriptomics.

  1. Park, C., Kim, S. B., Choi, S. H., & Kim, S. (2021). Comparison of 16S rRNA Gene Based Microbial Profiling Using Five Next-Generation Sequencers and Various Primers. Frontiers in microbiology, 12, 715500.
  2. Armstrong, A. J. S., Horton, D. B., Andrews, T., Greenberg, P., Roy, J., Gennaro, M. L., Carson, J. L., Panettieri, R. A., Barrett, E. S., & Blaser, M. J. (2023). Saliva microbiome in relation to SARS-CoV-2 infection in a prospective cohort of healthy US adults. EBioMedicine, 94, 104731.