How Does Third Generation Sequencing Work?
TGS technologies function by directly detecting the sequence of nucleotides in a single DNA or RNA molecule. SMRT sequencing uses zero-mode waveguides (ZMWs) to observe DNA polymerase activity in real-time, while ONT sequencing employs nanopores embedded in a synthetic membrane to detect changes in electrical conductivity as nucleotides pass through the pore.
Advantages of Third Generation Sequencing
Long-Read Capability: TGS can produce read lengths up to hundreds of kilobases, significantly longer than those achievable with earlier technologies. This is particularly useful for studying structural variations, complex genomic regions, and full-length RNA transcripts.
Real-Time Sequencing: TGS can provide results in real-time, which is crucial for rapid outbreak response and disease tracking.
Single-Molecule Resolution: The ability to sequence single molecules eliminates the need for PCR amplification, reducing bias and improving accuracy.
Epigenetic Modifications: TGS can detect epigenetic modifications such as DNA methylation directly, which is essential for understanding gene regulation and disease mechanisms.
Applications in Epidemiology
Pathogen Identification and Characterization
TGS enables the rapid and accurate identification of
pathogens during outbreaks. Long-read sequencing allows for the comprehensive characterization of viral, bacterial, and parasitic genomes, including regions that are difficult to sequence with short-read technologies. This facilitates the identification of novel strains and mutations that could affect virulence and transmissibility.
Antimicrobial Resistance
The ability to sequence entire genomes in a single read allows researchers to identify
antimicrobial resistance genes and their genomic context. This is crucial for tracking the spread of resistance and informing treatment strategies.
Surveillance and Monitoring
TGS can be used for ongoing
surveillance of pathogen populations, enabling the detection of emerging strains and monitoring of evolutionary changes. This is vital for understanding the dynamics of infectious diseases and implementing effective public health interventions.
Microbiome Studies
The human microbiome plays a significant role in health and disease. TGS facilitates the in-depth analysis of
microbial communities by providing long reads that can span entire operons or rRNA genes, offering more accurate taxonomic and functional profiling.
Challenges and Limitations
High Cost
The cost of TGS is currently higher than that of second generation sequencing, although it is expected to decrease as the technology matures and becomes more widespread.Error Rates
TGS technologies, particularly ONT, have higher raw error rates compared to second generation sequencing. However, these errors can be mitigated through bioinformatics approaches and consensus sequencing.
Data Analysis
The large volumes of data generated by TGS require advanced
bioinformatics tools and expertise to analyze. This can be a barrier for some research groups and public health laboratories.
Future Directions
The future of TGS in epidemiology looks promising, with ongoing advancements aimed at reducing costs, improving accuracy, and enhancing computational tools. The integration of TGS with other 'omics' technologies and real-time data sharing platforms will further enhance our ability to respond to infectious disease threats and improve public health outcomes.
