Shotgun Metagenomic Sequencing (SMS) vs Shallow Shotgun Metagenomic Sequencing (SSMS) Comparison

Metagenomics studies the entire microbial community in a specific habitat by recovering microbial genetic materials for sequencing without prior microbial isolation and culture.  

Microbial characterization has historically relied on 16S rRNA gene (16S) sequencing due to its accessibility and lower cost compared to shotgun metagenomic sequencing (SMS). However, 16S sequencing provides limited species-level resolution and does not offer a direct assessment of other taxa such as viruses and fungi, or functional gene content.

Deep shotgun metagenomics or Shotgun metagenomics sequencing (SMS) can elucidate the community structure and species classification, as well as provide insights into systemic evolution, gene function, metabolic network, and antibiotic resistance gene information. However, the deep insights with shotgun metagenomic sequencing come with a significantly higher cost than 16S sequencing. 

Sequencing at different sequencing depths allows for multiple applications and analysis purposes. Shallow shotgun metagenomic sequencing (SSMS) has emerged as an approach to bridge the gap between 16S sequencing and deep metagenomic sequencing. SSMS is cost-competitive with 16S sequencing, while also providing species-level resolution.

Comparisons between shotgun metagenomic sequencing approaches
Methodology Shotgun
Metagenomic Sequencing
Shallow Shotgun
Metagenomic Sequencing
Platform Type Illumina NovaSeq 6000
Read Length Paired-end 150 bp
Taxonomic Resolution Species-level
Taxonomic Coverage All taxa
Sequencing Depth
6-12 Gb
(>12 Gb may be recommended in certain cases)
1-3 Gb
Taxonomy Annotation Micro NR/ NR database Kraken 2
Analysis Method Assembly-based No assembly, K-mers
Standard Analysis
  • Microbial community characterization
  • Gene function annotation
  • Metabolic pathways
  • Antibiotic resistance gene annotation
  • Microbial community characterization
  • Sample Requirements 
    Sample Type Amount Volume Concentration Purity
    Genomic DNA ≥ 200ng ≥ 20 µ ≥ 10 ng/ µl OD260/280 = 1.8-2.0,
    No degradation,
    no contamination,
    no color
    Deep Shotgun Metagenomic Sequencing Shallow Shotgun Metagenomic Sequencing
    Pre-extracted DNA, soil, environmental water, stool, saliva, vaginal, oral, and rectal swabs. Pre-extracted DNA, stool, saliva, vaginal, oral, and rectal swabs.
  • High biomass with low-host contamination microbial samples is recommended for both services.
  • * For Shallow Shotgun Metagenomic Sequencing, we are currently only accepting human and animal-based samples in accordance with the current research available.
    Analysis Content Comparison
    Similarities in both analyses
    Hierarchical Cluster Heatmap
    The abundance distribution heatmap produces the dominant 35 genera of all samples.
    Sample Cluster Tree
    Visualize sample similarity according to the Bray-Curtis distance calculated from relative taxonomic abundance.
    LEfSe: Cladogram
    The cladogram diagram visualizes the significantly different microbial species within three groups. The nodes of the different colored groups represent the microbial species that play an essential role. The species classification at the phylum, class, order, family, and genus is shown from the inside to the outside.
    Analysis in deep shotgun metagenomic sequencing only
    Function Annotation Results
    Based on Unique gene annotation results, a cartogram of annotated gene numbers of each database was visualized.

    Antibiotic Resistance Gene Annotation
    Provides the relative abundance of different resistance genes after annotation with the ARDB database.

    Metabolic Pathway Analyses
    The metabolic pathway analyses provide visualization for both the annotated metabolic pathway and a comparison overview that showcases the common and unique pathway between groups or samples.
    Deciding which microbial application may be right for your microbiome research?

    About Novogene
    Novogene is a pioneer in applying cutting-edge molecular biology technology and high-performance computing to research in the fields of life science and human health. Our vision is to continue as a global leader in the delivery of genomics services and solutions. With one of the largest sequencing capacities in the world, we utilise our deep scientific knowledge, first-class customer service and unsurpassed data quality to help clients realise their research goals in the rapidly evolving world of genomics. Novogene is committed to become your trusted genomics partner.
    NovogeneAIT Genomics is a joint venture between Novogene and AIT Biotech focusing in Asia Pacific, Middle East, and Africa (AMEA) region. The AMEA regional headquarters of Novogene is in Singapore, overseeing a dynamic and diverse region with a presence in Singapore, Japan, Hong Kong SAR, Taiwan, Australia, India, the Middle East and across the region through our network of local partners.
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