In recent years, the landscape of sequencing technologies has undergone a transformative shift, transitioning from research settings to the realm of clinical laboratories. This transformation has been catalyzed by rapid technological advancements and significant cost reductions. Despite a multitude of microorganisms known to inflict human infections, prevailing diagnostic techniques merely scratch the surface, leaving a vast array of pathogens unidentified. This persistent challenge is further exacerbated by the continuous emergence of novel pathogens. Enter metagenomic next-generation sequencing (mNGS), an all-encompassing and precise approach for microbial detection and taxonomic characterization. While numerous studies and case reports underscore the efficacy of mNGS in enhancing the diagnosis, treatment, and tracking of infectious diseases, formidable obstacles still lie on the path forward.

Unleashing the Power of NGS

The evolution of sequencing technologies from first-generation methods to the sophisticated realm of next-generation sequencing technologies, also commonly known as NGS or high-throughput sequencing, has effectively mitigated the low-throughput challenges that plagued the initial techniques. Since the year 2000, the advent of massively parallel sequencing technologies has orchestrated a revolution in high-throughput sequencing capabilities. Critical breakthroughs, including pyrophosphate sequencing, reversible terminator chemical sequencing, and supported oligonucleotide ligation and sequencing, have catalyzed a monumental increase in throughput potential. Remarkably, this progression has unfolded alongside a dramatic reduction in sequencing costs, further democratizing access to these cutting-edge tools.

The Promise of Long-Read Sequencing

The long-read sequencing technologies (Nanopore and PacBio Sequencing), alternatively referred to as single-molecule sequencing, stand as a testament to innovation. In contrast to NGS, these technologies boast the ability to detect a staggering ten nucleotides per second, culminating in a substantial reduction in sequencing time. Beyond speed, these technologies unlock the potential to deduce full-length mRNA sequences through their ultra-long read lengths. An extraordinary attribute of long-read sequencers is their ability to directly sequence raw DNA/RNA samples, obviating the need for PCR amplification. Additionally, they exhibit an unbiased approach toward CG nucleotides and offer the direct detection and retrieval of methylation information. However, despite these remarkable advantages, the integration of long-read sequencing into clinical applications remains limited due to its elevated error rate and the associated financial constraints.

Navigating the Path from Wet Lab to Dry Lab

The high-throughput sequencing of pathogens necessitates a two-fold process: wet lab experimentation and subsequent bioinformatics analysis. Wet lab endeavors encompass sample pretreatment, nucleic acid extraction, library construction, and ultimately, sequencing. Meanwhile, dry lab bioinformatics analysis encompasses tasks like data quality control, the elimination of human sequences, comparison of microbial species sequences, and analysis of drug resistance or virulence genes.

Unveiling the Process: Key Stages

• Sample Collection: Precision in collecting samples from the primary infection site augments detection rates substantially.
• Sample Pre-Treatment: Different samples necessitate distinct pre-treatment strategies—liquefaction for sputum, dewaxing for FFPE samples, and homogenization for tissues. Techniques like filtration, differential centrifugation, DNA enzyme hydrolysis, and methylation reagent treatment mitigate the proportion of human DNA in the samples.
• Nucleic Acid Extraction: Different protocols are required for DNA and RNA extraction.
• Library Construction: The choice of library construction methods aligns with the sequencing platform and intended purpose.
• Sequencing: Leading next-generation sequencing platforms, like those offered by Illumina and UW Genetics, dominate this stage.
• Bioinformatic Analysis: Raw data analysis lays the foundation for subsequent species and antibiotic resistance gene identification.
• Reporting: Analysis outcomes facilitate the identification of potential pathogens.

NGS: Revolutionizing Pathogen Identification

Whole-genome next-generation sequencing stands as a beacon of progress, enabling swift, comprehensive identification of bacteria, fungi, viruses, and parasites. This approach eliminates the need for a priori assumptions about the causative agent, delivering unparalleled sensitivity and accuracy compared to conventional culture-based methods. mNGS proves particularly potent in identifying mycobacteria, anaerobes, atypical pathogens, and viruses, demonstrating resilience even in the presence of antibiotics. This potential for precise diagnosis and differential diagnosis of infectious diseases has garnered validation through an array of studies.

In Conclusion

The journey of sequencing technologies from the corridors of research to the forefront of clinical diagnostics has been propelled by technological leaps and cost reductions. Metagenomic next-generation sequencing (mNGS) represents a paradigm shift, offering a holistic and dependable strategy for microbial detection and taxonomic profiling. While the accomplishments in leveraging mNGS for disease diagnosis and management are notable, formidable challenges still beckon for resolution. Through the unrelenting pursuit of precision, clinical microbiology stands poised to redefine infectious disease diagnostics and treatment.