Shotgun metagenomic sequencing

Shotgun metagenomic sequencing has three basic steps: library preparation, sequencing, and analysis. Extracted DNA is randomly sheared, adapters are added, and the library is sequenced on an NGS system. Reads are then classified taxonomically and analyzed for gene content using bioinformatics pipelines such as the DRAGEN Metagenomics pipeline. Multiple samples can be multiplexed in a single run to sequence thousands of organisms in parallel.

After sequencing, reads are classified taxonomically and assessed for community composition and gene content. Analysis can include quality control, host-read filtering, organism detection, and single-sample or aggregate reporting across a study. The DRAGEN Metagenomics pipeline performs taxonomic classification of reads and provides single-sample and aggregate reporting for shotgun metagenomics workflows.

16S rRNA sequencing is an amplicon-based method that targets a single taxonomic marker in bacteria and archaea. Shotgun metagenomic sequencing captures genomic DNA from virtually all organisms in a sample, providing broader taxonomic coverage and access to functional gene information. Use 16S rRNA sequencing to profile bacterial communities cost-effectively. Use shotgun metagenomics when you need species-level resolution, functional insight, or detection of fungi, viruses, and genes such as antimicrobial resistance markers.

Shallow shotgun metagenomic sequencing uses lower read depth than traditional deep shotgun metagenomics while still sequencing whole-community DNA rather than a single amplicon. It can offer higher discriminatory power and reproducibility than 16S sequencing at a lower cost and complexity than deep shotgun sequencing. It is a practical option when you need species-level characterization or functional profiling without the read depth required for the most sensitive detection targets.

Not always. The sequencing depth you need depends on the question you are asking. Taxonomic profiling of abundant community members typically requires far fewer reads than detecting low-abundance organisms or profiling antimicrobial resistance gene families. Rarefaction curves can help estimate whether additional sequencing will reveal more species in your sample. Shallow shotgun approaches can be a useful middle ground when deep sequencing is not required for your study goals.

Researchers use metagenomic sequencing to study microbial communities in human, environmental, and agricultural samples without culturing each organism. Common uses include identifying which organisms are present in a sample, estimating relative abundance, surveying functional gene content, supporting pathogen and outbreak research, and monitoring antimicrobial resistance. For more information, visit human microbiome analysis, environmental metagenomics, and antimicrobial resistance surveillance.