Detection of Microbial Pathogens: mNGS or tNGS

Metagenomic Next-Generation Sequencing (mNGS)

Metagenomic Next-Generation Sequencing (mNGS), also known as environmental genomics or community genomics, represents an advanced methodology for analyzing the genomes of microbial communities. This technique involves the direct extraction of the entire DNA/RNA content of microorganisms from a sample, followed by high-throughput sequencing to elucidate the genomic composition and functions of the microbial community. Unlike traditional methods, mNGS obviates the need for isolation and culture of microorganisms, thereby providing a more comprehensive view of the microbial community structure and functions.

In the domain of pathogen detection, mNGS plays a crucial role. As an unbiased sequencing approach, mNGS is theoretically capable of identifying all pathogens present within a sample. It facilitates the rapid and objective detection of various pathogenic microorganisms in clinical samples, including viruses, bacteria, fungi, mycoplasma, chlamydia, and parasites, as well as resistance genes and virulence factors. By identifying microbial markers associated with diseases, mNGS offers novel insights into disease prevention and treatment.

The application of mNGS is particularly advantageous for diagnosing critical and complex infections, significantly enhancing pathogen detection rates, especially for rare and atypical pathogens. Furthermore, mNGS demonstrates the capacity to detect emerging and uncommon pathogens, and it exhibits clear advantages in the pathogen detection of mixed infections.

Figure 1: Diagram showing the steps of the metagenomic analysis workflow, including sample collection, DNA extraction, and sequencing. Figure 1. Overview of Metagenomic Analysis Workflow

Advantages of Metagenomics:

Diverse Sample Detection: Metagenomics permits the direct extraction of total DNA from microbial communities without the need for isolation and culture of individual microorganisms.
Broad Detection Spectrum: This approach enables the simultaneous identification of nearly all microbial species present within a sample.
Comprehensive Data Analysis: In addition to species identification, metagenomics provides significant insights into the expression of key genes within the community.
Rapid and Efficient Analysis: Metagenomics facilitates the quick, efficient, and large-scale analysis of microbial communities.

Figure 2: Schematic of metagenomic data analysis workflow, highlighting data processing, quality control, and functional annotation. Figure 2. Metagenomic data analysis workflow

Services you may interested in
Pathogen Sequencing Solutions	Pathogens mNGS Sequencing Service
Pathogen Targeted Sequencing (tNGS)

Host DNA Removal

To enhance the study and analysis of non-host microbial genomic information and to gain deeper insights into microbial functions and interactions, it is essential to address the contamination of host DNA. This contamination is unavoidable in eukaryotic host samples, particularly those with low microbial content and high host DNA content, such as sputum or dust. Such contamination can significantly compromise the depth and accuracy of metagenomic sequencing. Therefore, the necessity of host DNA removal should be evaluated prior to sequencing.

Targeted Next-Generation Sequencing (tNGS)

tNGS is a high-throughput sequencing technique that focuses on specific genes or genomic regions. By sequencing only targeted gene sequences, tNGS enhances detection sensitivity and eliminates interference from host nucleic acids. This method primarily detects known pathogenic microorganisms and their resistance genes, ranging from dozens to hundreds within a sample.

Figure 3: Illustration of two target enrichment approaches for NGS, depicting principles of PCR and hybridization probe methods. Figure 3. Target Enrichment Approaches for NGS. The underlying principles of the two prominent target enrichment technologies are depicted schematically.

Enrichment Strategies in tNGS

tNGS employs two principal enrichment strategies based on the enrichment approach: PCR amplicon enrichment and hybrid capture probe enrichment.

PCR Amplicon Enrichment: This technique involves the use of primers complementary to known nucleotide sequences to PCR amplify viral genomes spanning hundreds to thousands of base pairs prior to NGS sequencing. This method is particularly useful for enriching small viral genomes.

Hybrid Capture Probe Enrichment: In this approach, small RNA/DNA probes are designed to be complementary to reference sequences of pathogens. Unlike the specific PCR amplicon-based method, probe-based hybrid capture allows the entire genome to be covered by overlapping probes. These probes engage in hybridization reactions to capture complementary DNA sequences bound to a solid phase.

Advantages of tNGS

Specific Enrichment: tNGS specifically captures and enriches target pathogenic microorganisms, notably enhancing virus diagnostics without the need for culturing.

Enhanced Sensitivity: Compared to mNGS, tNGS significantly increases detection sensitivity in samples with high host genomic content.

Cost Efficiency: By sequencing only the genes of interest, tNGS offers a more cost-effective solution.

Aspect	Hybrid Capture Enrichment	PCR Amplicon Enrichment
Enrichment Principle	Uses single-stranded DNA or RNA probes complementary to the genomic regions of interest for gene hybrid capture enrichment.	Utilizes sequence-specific primers flanking the genomic regions of interest for PCR-based amplification.
Gene Targets per Panel	No limitation on the number of gene targets; preferred method for large panels.	Typically used for a smaller number of gene targets, limited by primer design.
Consistency of Enrichment	Higher consistency in target region enrichment, lower sequencing failure rate.	Lower uniformity of target enrichment, higher sequencing failure rate.
Workflow Time	Relatively longer.	Relatively shorter.
Applicability	Suitable for large genomes or targeted sequencing.	Suitable for smaller target regions.
Applicable Sample Types	DNA.	DNA and RNA.

References

Quince, C., Walker, A., Simpson, J. et al. Shotgun metagenomics, from sampling to analysis. Nat Biotechnol 35, 833–844 (2017).
Singh, R.R. Target Enrichment Approaches for Next-Generation Sequencing Applications in Oncology. Diagnostics 2022, 12, 1539.