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What’s the Difference Between Amplicon Next Generation Sequencing and Metagenomic Shotgun Sequencing?

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With the advent of next generation sequencing, scientists have at their disposal powerful tools that lets them run high-volume DNA sequencing in massively parallel processing systems quickly and at lower costs than ever before.

It’s a versatile technology that allows for a wide range of work in genomics, for disease detection and the development of therapeutics, to broadening our understanding of the way microorganisms attack people or how people’s health is affected when they have different mutations in their DNA.

As the gene-sequencing technology matures and evolves, there are different approaches to sequencing for technicians to employ in laboratories, clinics, research facilities, and the like.

For example, it’s useful to become familiar with the difference between amplicon next generation sequencing and metagenomic shotgun sequencing.

About Amplicon Next Generation Sequencing

Next generation sequencing technology based on amplicons enables technicians to conduct metataxonomic detection quickly and accurately, at rates their laboratory managers can more readily afford, as noted by CD Genomics. Amplicon-based NGS allows for metataxonomic amplicon profiling based on ribosomal RNA with variable and conservative regions to supply the needed genetic material in experiments.

It relies on oligonucleotide probes which home in on and capture the region you are interested in. Then, these captured areas are amplified with NGS to identify the genetic information.

Researchers use this approach to do taxonomic and phylogenetic studies on archaea and bacteria. Individuals studying protists and fungi or who are doing work on fungal ecology are interested in their molecular phylogeny, to species and genus level identification.

Research objectives of those doing work with amplicon-based NGS efforts are typically looking at phylogenetic relationships between species, the biodiversity of a community of microorganisms, or what species are in a sample.

You can use this technology quite effectively for identifying microorganisms at the genus level (some species-level identification is also possible with this technology).

About Metagenomic Shotgun Sequencing

To bypass some limitations of amplicon-based next generation sequencing, metagenomic shotgun sequencing can be used. You are not focusing on targeted regions, as is the case with amplicon NGS. Instead, akin to spreading a wide random “shotgun” pattern, you are independently sequencing every DNA fragment in the sample.

It involves shearing DNA into tiny pieces, then applying a universal primer at the fragment ends so scientists can amplify with PCR for sequencing.

The system will make some reads, identifying taxonomically useful regions along the lines of 18S and 16S, per CD Genomics.

Sometimes you will be obtaining reads from coding sequences that show you details about biological functions encoded in the organism’s genome. This, in turn, helps researchers learn more about the functions and biodiversity present in a given community of microbes.

Research objectives for metagenomic shotgun sequencing work often include taxonomic analysis (as is the case with amplicon NGS) as well as detailed research into genes and microbial community functions (for example, scientists investigating KEGG or pathway analysis).

You will use metagenomic shotgun sequencing to determine microbes on a species-by-species basis. Sometimes, you will be able to distinguish strains and subspecies in a sample. It’s a prized method for detecting novel genes in the laboratory at high resolution. Researchers will turn to this version of sequencing when they want to obtain all information in the genes of viruses, eukaryotes, and prokaryotes.

Different Approaches for Different Scientific Objectives

It’s encouraging to consider that technicians can work with such varying systems as amplicon next generation sequencing and metagenomic shotgun sequencing.

Both approaches happen to rely on the improved technology and computational capabilities that had to be invented to make massively parallel sequencing and high bandwidth data transmission and storage systems possible. It certainly pays to keep an eye on technology developments as much as on innovations in the life sciences these technology updates help to support.

Agan Jarick
the authorAgan Jarick