Microbial Genomics
Microbial Genomics

Shotgun metagenomic sequencing

Sequence thousands of organisms in parallel using NGS

What is shotgun metagenomic sequencing?

Shotgun metagenomic sequencing helps allow researchers to comprehensively sample all genes in all organisms present in a given complex sample.1,2 This next-generation sequencing (NGS) method enables microbiologists to evaluate bacterial diversity and detect the abundance of microbes in various environments. Shotgun metagenomics also provides a means to study unculturable microorganisms that are otherwise difficult or impossible to analyze.3

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Why sequencing depth is important for shotgun metagenomics

Sequencing depth refers to the number of sequencing reads that align to a reference region in a genome. This is important because a greater sequencing depth provides stronger evidence that the results are correct. Although shotgun metagenomic sequencing is comprehensive, this approach can be costly and complex to analyze compared to 16S sequencing. Shallow shotgun sequencing is a method that provides a helpful alternative against the drawbacks of both methods, providing shallower reads compared to full shotgun sequencing while enabling higher discriminatory and reproducible results compared to 16S sequencing.4

Applications of shotgun metagenomic sequencing

Human microbiome analysis

See how next-generation sequencing (NGS) can empower studies capable of surveying the genomes of entire microbial communities, including those of unculturable organisms.

Genomic surveillance

Learn how researchers use genomic surveillance to study pathogens at a molecular level, providing insights into their behavior and evolution in populations around the world.

Recommended shotgun metagenomics workflows

1
Prep
For 2 × 150 bp runs only:

Illumina DNA Prep

Fast, all‑purpose tagmentation workflow supporting a wide range of DNA inputs (1–500 ng) for whole-genome sequencing (WGS) and microbial sequencing.

Illumina DNA PCR-Free Prep

PCR‑free workflow delivering highly uniform WGS results and improved accuracy for sensitive microbial applications.

Nextera XT DNA Library Preparation Kit

Ultra‑fast amplicon and small‑genome prep optimized for low DNA input (1 ng) and high‑plex multiplexing.

Compatible with 2 × 150 and 2 × 300 bp runs:

TruSeq DNA Nano (550 bp average insert size)

Low‑input (~100 ng) library prep offering reliable coverage uniformity and reduced bias across genomes.

TruSeq DNA PCR-Free (550 bp average insert size)

A simple, gel‑free, PCR‑free WGS prep that provides accurate, comprehensive coverage of complex genomes with scalable throughput.

2
Sequence

MiSeq i100 Series

The MiSeq i100 Series delivers our fastest run times yet, breakthrough simplicity, and significant sustainability advancements to empower every lab.

NextSeq 550 System

Combine proven NGS technology with tunable output to enable sequencing and array capabilities across a range of applications. 

NextSeq 1000 and 2000 Systems

Empower discoveries with a comprehensive workflow from start to finish.

3
Analyze

DRAGEN Metagenomics Pipeline

The DRAGEN Metagenomics pipeline performs taxonomic classification of reads and provides single sample and aggregate reporting.

1
Prep
For 2 × 150 bp runs only:

Illumina DNA Prep

Fast, all‑purpose tagmentation workflow supporting a wide range of DNA inputs (1–500 ng) for whole-genome sequencing (WGS) and microbial sequencing.

Illumina DNA PCR-Free Prep

PCR‑free workflow delivering highly uniform WGS results and improved accuracy for sensitive microbial applications.

Nextera XT DNA Library Preparation Kit

Ultra‑fast amplicon and small‑genome prep optimized for low DNA input (1 ng) and high‑plex multiplexing.

Compatible with 2 × 150 and 2 × 300 bp runs:

TruSeq DNA Nano (550 bp average insert size)

Low‑input (~100 ng) library prep offering reliable coverage uniformity and reduced bias across genomes.

TruSeq DNA PCR-Free (550 bp average insert size)

A simple, gel‑free, PCR‑free WGS prep that provides accurate, comprehensive coverage of complex genomes with scalable throughput.

2
Sequence

NextSeq 1000 and 2000 Systems

Empower discoveries with a comprehensive workflow from start to finish.

NovaSeq 6000 System

A robust and scalable system designed to adapt to your needs and bring discoveries within reach.

NovaSeq X Series

Achieve extraordinary throughput and accuracy to perform data-intensive applications at production scale.

3
Analyze

DRAGEN Metagenomics Pipeline

The DRAGEN Metagenomics pipeline performs taxonomic classification of reads and provides single sample and aggregate reporting.

Guide to shotgun metagenomic sequencing

Read this guide to understand how to sequence complex microbial samples for important diseases or to gain insight into community-level microbial biodiversity and function.

Two researchers, man and woman, kneeling in the middle of a forest near at the bed of a river, holding a jar filled with murky river water, looking closely at the composition and assessing.

Related methods

16S and ITS rRNA sequencing

NGS-based Internal Transcribed Spacer (ITS) and 16S rRNA gene sequencing are well-established methods for comparing sample phylogeny and taxonomy from complex microbiomes or environments that are difficult or impossible to study.

Metatranscriptomics sequencing

Learn about how to collectively study expressed mRNA from complex microbial communities. This method also allows researchers to gain important insights on how microbes respond to a given environment at a specific time.

Additional resources

The mysterious world of microbes

Read how researchers are investigating the genomes of microbes to improve our understanding of human health, disease, and microbial evolution.

The time is now for microbiome studies

In this interview, Dr. Joseph Petrosino, PhD, Director of the CMMR and Chief Scientific Officer of Diversigen, discusses how he uses whole-genome shotgun sequencing and transcriptomics to refine drug discovery and development.

Soil metagenomics workflow

Read this Application Note to learn more about using shotgun metagenomics for scalable studies to study soil microbes.

Metagenomic sequencing on the NovaSeq 6000 System

In this Application Note, see how shotgun metagenomics is used to study a diverse array of bacterial and metagenomic samples with the NovaSeq 6000 System for high-throughput workflows with longer read lengths.

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References

  1. Quince, C., Walker, A., Simpson, J. et al. Shotgun metagenomics, from sampling to analysis. Nat Biotechnol. 2017; 35, 833–844. doi: 10.1038/nbt.3935
  2. New FN, Brito IL. What Is Metagenomics Teaching Us, and What Is Missed? Annu Rev Microbiol. 2020; 74:117-135. doi: 10.1146/annurev-micro-012520-072314.
  3. Sharpton TJ. An introduction to the analysis of shotgun metagenomic data. Front Plant Sci. 2014; Jun 16;5:209. doi: 10.3389/fpls.2014.00209
  4. Hillmann B, Al-Ghalith GA, et al. Evaluating the Information Content of Shallow Shotgun Metagenomics. mSystems. 2018 Nov 13;3(6):e00069-18. doi: 10.1128/mSystems.00069-18.