16S rRNA Sequencing VS Shotgun Sequencing for Microbial Research

16S rRNA Sequencing

16S rRNA gene sequencing is a kind of amplicon sequencing that targets and reads an area of the 16S rRNA gene, which is found in all Bacteria and Archaea, so it can only define these microorganisms. Other kinds of amplicon sequencing, like ITS sequencing for fungi or 18S sequencing for protists, can distinguish various microorganisms.

The result of 16S rRNA sequencing is sequencing 'reads' (DNA sequence strings) that can be analyzed using a number of basic bioinformatic processes, which are referred to as 'pipelines' when they are integrated. These bioinformatic pipelines 'clean' the data by removing sequencing errors or questionable reads, which can then be associated with microbial genomic databases to correctly determine and profile the bacteria (and archaea) that were visible in the specimens.

Shotgun Metagenomic Sequencing

Shotgun metagenomic sequencing entails breaking ('fragmenting') DNA into many tiny chunks at random, similar to how a shotgun would tear something up into many pieces. The DNA sequences of these fragmented pieces of DNA are then stitched back together using bioinformatics to recognize the species and genes existing in the specimen. Shotgun metagenomic sequencing, unlike 16S rRNA sequencing, can read all genomic DNA in a specimen rather than just one particular area. Shotgun sequencing can simultaneously identify and profile bacteria, fungi, viruses, and a variety of other microorganisms, which is useful for microbiome research. As genomes are sequenced, microbial genes observed in the specimen (the metagenome) can be identified and profiled, providing additional information about the microbiome's functional potential. When compared to 16S rRNA sequencing, metagenomic sequencing requires a few extra steps.

To evaluate the outcomes of shotgun sequencing reads, more complicated bioinformatics techniques are utilized. Quality filtering steps are also included in shotgun metagenomics bioinformatics pipelines, after which the cleaned sequencing data can be compiled to generate partial or full microbial genomes or aligned to databases of microbial marker genes. Many of these pipelines now include online tutorials and user interfaces to help people who aren't experts in bioinformatics with their analyses. The findings include information on the relative abundances of bacteria, fungi, viruses, and other microbes in the specimen, as well as curated lists of microbial genes.

16s rRNA VS Shotgun Metagenomic Sequencing

Factors 16s rRNA Sequencing Shotgun Metagenomic Sequencing
Functional profiling (profile microbial genes) No (although 'predicted' functional profiling is an option). Yes, indeed (but it only reveals information on functional potential)
Taxonomic resolution: Genus, species, strain Bacterial genus (or species), depending on location (s) centered Species of bacteria (sometimes strains and single nucleotide variants, if sequencing is deep enough)
Taxonomic coverage Bacteria and archaea are two types of bacteria. Viruses are included in all taxa.
Bioinformatics requirements Expertise ranging from beginner to intermediate Intermediate to advanced level of knowledge
Databases Established and carefully curated Comparatively new, still improving
Sensitivity to host DNA contamination Low (but PCR success according on the absence of inhibitors and the presence of a detectable microbiome) High, but differs depending on the sample kind(but this can be mitigated by calibrating the sequencing depth)
Bias High-to-medium (acquired taxonomic composition is dependent on selected primers and targeted variable area) Lower (while metagenomics is “untargeted,” experimental and analytical biases can be proposed at any point)


  1. Durazzi F, Sala C, Castellani G, et al. Comparison between 16S rRNA and shotgun sequencing data for the taxonomic characterization of the gut microbiota. Scientific reports.2021 Feb 4;11(1).
  2. Mori H, Maruyama T, Yano M, et al. VITCOMIC2: visualization tool for the phylogenetic composition of microbial communities based on 16S rRNA gene amplicons and metagenomic shotgun sequencing. BMC systems biology. 2018 Mar;12(2).
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