How Shotgun Metagenomic Sequencing Differs from 16S rRNA Gene Sequencing

Posted by Vivian creative on August 4th, 2021

It's suggested by microbiology researchers that 16S rRNA sequencing and shotgun metagenomic sequencing are advanced tools for metagenomics studies, be it microbial diversity characterizationor the distinction of pathogenic and commensal bacteria. Metagenome sequencing and genome assembly will advance the development of live biotherapeutic products for various applications, including diagnosis, treatment, mitigation, and prevention of diseases.

16S rRNA gene sequencing and shotgun metagenomic sequencing are common means to acquire data for microbial profiling and metagenomics analyses. However, when arranging a new investigation, microbiome researchers could face a dilemma of which to choose as both are frequent methods in metagenomics studies. This post introduces and compares these two methods to show their differences, advantages, shortcomings, and other features.

Shotgun Metagenomic Sequencing

Metagenomics studies the functional genomes of microbial communities and analyzes the genomics of microbial DNA from environmental communities. Shotgun metagenomic sequencing is a new environmental sequencing way that can analyze whole microbial communities in various environments for taxonomic profiling and functional analysis of microbial communities.

The ability to examine thousands of organisms in parallel and comprehensively sample all genes makes shotgun metagenomic sequencing the next-generation sequencing (NGS) for researchers better understanding community biodiversity and functions.

16S rRNA Gene Sequencing

16S rRNA gene sequencing, also known as 16S sequencing, is a common amplicon sequencing method that identifies and compares bacteria or fungi present within a given sample. This approach is based on the 16S rRNA gene, a taxonomic genomic marker existing in almost all bacteria and archaea. 16S rRNA gene sequencing allows researchers to check the genetic diversity and microbial composition in a sample, meanwhile generating a taxonomy profile after classifying genus and species in different microbial populations.

Community analysis using next-generation sequencing allows the rapid sequencing of the base pairs in DNA or RNA samples, so NGS-based 16S rRNA sequencing is also developed to extract DNA, prepare genomes, and build 16S rDNA libraries.

What is the difference?

Though both are commonly used in metagenomics studies, shotgun metagenomic sequencing differs from 16S rRNA gene sequencing in some ways.

Being completely accurate, traditional 16S sequencing is not for metagenomics. Metagenomics focuses on studying the collective genomes of microbial communities but 16S sequencing only targets the diversity of a single ribosomal gene, 16S rRNA. Therefore, the whole metagenomic profiling using shotgun sequencing is unique, including taxonomic profiling and functional analysis.

As a result, shotgun metagenomic sequencing can detect members with a low abundance in the microbial communities while 16S sequencing is prone to miss certain viruses, fungi, or low-abundance members.

Which is better?

Actually, using which methods largely depends on the main purpose of the investigation and the budget. For instance, metabolic pathway analysis that is beyond taxonomy profiling will get wider genomic coverage using shotgun metagenomic sequencing. And for composition profiling provided with limited funding, 16S sequencing is a better choice.

Moreover, current 16S databases cover more than whole-genome databases on the condition that there are more studies on the whole genomes of microbes about human microbiomes than other environments. Shotgun metagenomic sequencing is highly dependent on the reference database, meaning shotgun metagenomic sequencing is better for human-microbiome-related sample analysis with higher resolution and accuracy since the database is off the shelf.

The following are the pros and cons of shotgun metagenomic sequencing and 16S sequencing.

Advantages of shotgun metagenomic sequencing

• Fast sequencing process.
• Better taxonomy resolution, functional profiling, and cross-domain coverage.
• Less susceptible to the biases inherent in targeted gene amplification.
• More accurate in examining relative abundances of different organisms.

Disadvantages of shotgun metagenomic sequencing

• Sophisticated software and computing techniques in shotgun sequence assembly.
• More possible to have false positive errors.
• Less sample origin compatibility
• Higher price

Advantages of 16S sequencing

• Universal distribution and higher abundance of target gene
• Easier measurements of phylogenetic relationships across different taxa
• Less affected by Horizontal gene transfer
• More affordable costs

Disadvantages of 16S sequencing

• Too low resolution to differentiate between closely related species.
• Less reliable relative abundance due to amplification biases.

Conclusion

Shotgun metagenomic sequencing and 16S rRNA gene sequencing are both widely applied in microbial identification services and projects, but with main differences in the budget, sample types, purpose of analyses, taxonomy resolution, and sequencing principle. Consequently, these factors decide which is better for the microbiome research project. Thanks to the drop in sequencing costs, characterizations of microbiomes are gradually moving from 16S rRNA gene sequencing to shotgun metagenomics sequencing.