Metagenomics

Shotgun sequencing and amplicon sequencing are two widely used metagenomics approaches in microbiome research which allow detection and quantification of specific microbes in samples. Shotgun sequencing and long-read sequencing facilitate de-novo discovery of novel microbial species and viruses.

We offer both shotgun and amplicon sequencing services. For any study, the choice of sequencing technique will depend on the sample type, the study design, and the aim of the study. 

Shotgun metagenomic sequencing involves the sequencing of all DNA present in a sample. The microbial DNA is randomly broken into small fragments, which are sequenced, and mapped to our reference catalogues (e. g. our human microbiome reference) or assembled based on sequence overlap and binned into genomes. Shotgun data allows for the most comprehensive and highest resolution analysis of the microbiome

 

What are the advantages and limitations of shotgun sequencing? Advantages of shotgun sequencing: - Unbiased analysis of microbial communities, - High taxonomic resolution, allowing profiling of Bacteria, Archaea, Eukaryotes, viruses and bacteriophages, - Powerful for de-novo discovery and profiling of clonal populations or strains, and for source tracking using single nucleotide variant (SNV) information, - Yields functional information about the microbial community and strains, - Allows for antibiotic resistance gene analysis, - Enables Metagenome-Assembled Genome (MAG) generation. Limitations of shotgun sequencing: - May not be ideal for some sample types (e. g. those with very-low microbial biomass, and high host contamination, like tissue biopsies), - Requires significant computational power and know-how.

Advantages and limitations of shotgun sequencing.

 

Our CSO, H. Bjørn Nielsen has pioneered the de novo identification and microbiome profiling using shotgun metagenomic data, and in collaboration with many international researchers, he contributed to a series of significant advances in microbiome profiling and analysis. ¹

Amplicon sequencing is a targeted approach that focuses on sequencing a specific region of the microbial genome – most commonly, the 16S ribosomal RNA gene, which is present in all bacteria and archaea. The amplified region is typically short, at around 300-600 base pairs, and since it varies in different taxonomic groups it allows for the classification of bacteria at the genus and sometimes species level.  

The mycobiota, the fungal gut microbiota, can be profiled by targeting the internal transcribed spacer 2 (ITS2) region of the fungal genome.

 

What are the advantages and limitations of amplicon sequencing? Advantages of amplicon sequencing (16S, ITS, 18S, SLS): - Quick turnaround time, - Cost-effective, - High-throughput, - Good for low-biomass samples. Limitations of amplicon sequencing (16S, ITS, 18S, SLS): - Limited taxonomic resolution (i.e. no information on strains), - Need for multiple amplicon methods to detect all types of microbes (Bacteria, Archaea and fungi), - Does not profile viruses or phages, - Cannot provide functional information.

Advantages and limitations of amplicon sequencing (16S, ITS, 18S, SLS).

 

 

Read more about our bioinformatic tools for maximizing insights from your data or contact us to discuss which microbiome analysis technique is better suited to your unique study. 

 

References

¹ Nielsen, H., Almeida, M., Juncker, A. et al. Identification and assembly of genomes and genetic elements in complex metagenomic samples without using reference genomes. Nature Biotechnology 32, 822–828 (2014). https://doi.org/10.1038/nbt.2939

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