How Long-Read Sequencing Reveals Hidden Genomic Secrets
The tiny world of microbes holds clues to solving humanity's biggest challenges in health, agriculture, and environmental sustainability.
Deep within every scoop of soil, every drop of seawater, and every human gut exist trillions of microorganisms—an invisible universe holding profound secrets about life itself. For centuries, studying these microbes was painstaking work, like assembling a complex jigsaw puzzle with most pieces missing. Today, long-read sequencing technologies are revolutionizing microbial genomics, allowing scientists to read entire microbial genomes with unprecedented completeness and accuracy. This technological leap is uncovering new insights into antibiotic resistance, environmental sustainability, and human health—one genome at a time.
Imagine trying to reconstruct a shredded letter by painstakingly matching torn edges. Until recently, this was essentially what scientists faced when sequencing microbial genomes. They would break DNA into tiny fragments, sequence them, and then computationally reassemble them.
The limitation was technology: short-read sequencing produced DNA snippets just a few hundred letters long, making it nearly impossible to correctly assemble repetitive regions or complex genomic structures. Like trying to reassemble a book from sentence fragments rather than paragraphs, crucial context was often lost.
Long-read sequencing technologies from Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have transformed this process by generating DNA reads tens of thousands of base pairs long—sometimes even exceeding 100,000 bases. This revolutionary approach provides the genomic equivalent of having entire chapters of a book rather than scattered sentences, making assembly far more accurate and complete.
The completeness and accuracy of microbial genome assembly directly impacts what scientists can discover:
Antibiotic resistance genes often reside in repetitive or complex genomic regions that were previously difficult to sequence.
Virulence factors that enable pathogens to cause disease can be identified more completely.
Metabolic pathways can be reconstructed in their entirety, revealing how microbes interact with their environment.
Evolutionary relationships become clearer when complete genomes rather than fragments are compared.
While long-read sequencing technologies have existed for several years, their use has required advanced computational skills and significant infrastructure—barriers that prevented many microbiologists from leveraging their power. The Italian node of the Microbial Resource Research Infrastructure (MIRRI-IT) has developed an innovative bioinformatics platform specifically designed to overcome these challenges 1 .
This service provides a complete analysis pipeline for long-read microbial sequencing data, supporting both prokaryotic and eukaryotic organisms through an integrated workflow that includes:
Upload long-read sequencing data from PacBio or Oxford Nanopore platforms
Multiple assemblers run in parallel (Canu, Flye, wtdbg2)
Evaluate assemblies using N50, L50, and BUSCO metrics
BRAKER3 for eukaryotes, Prokka for prokaryotes
InterProScan for protein domain analysis and functional assignment
Comprehensive report with downloadable results and visualizations
Three innovative aspects make this platform particularly valuable to the research community 1 :
An intuitive web interface allows researchers without bioinformatics expertise to set up and execute complex analyses.
The service transparently leverages powerful computing infrastructure to accelerate analysis.
Built on Common Workflow Language (CWL) and containerized with Docker, the pipeline ensures complete transparency and portability.
This combination of accessibility and computational power represents a significant advancement toward what the developers describe as "a user-centered scalable bioinformatics service for microbial research" 1 .
The platform supports data from the two leading long-read sequencing technologies 7 :
Uses circular consensus sequencing to achieve accuracies up to 99.9%
High Accuracy Long Reads CCS TechnologyMeasures changes in electrical current as DNA passes through nanopores, with recent flow cells achieving 99.5% accuracy
Real-time Sequencing Portable Direct RNA SequencingThe platform integrates cutting-edge bioinformatics tools at each analysis stage 1 :
| Analysis Stage | Tool | Function |
|---|---|---|
| Genome Assembly | Canu, Flye, wtdbg2 | Reconstruct complete genomes from sequence reads |
| Gene Prediction | BRAKER3 (eukaryotes), Prokka (prokaryotes) | Identify gene locations in assembled genomes |
| Functional Annotation | InterProScan | Determine protein functions based on domain analysis |
| Quality Assessment | BUSCO | Evaluate assembly completeness using universal orthologs |
To demonstrate the platform's capabilities, researchers selected three microorganisms of clinical and environmental significance from the TUCC culture collections 1 :
A fungus with environmental and clinical relevance
A clinically important bacterium known for antibiotic resistance
An emerging multidrug-resistant fungal pathogen
The research team processed each microorganism through the complete pipeline 1 :
The platform successfully generated high-quality assemblies and annotations for all three test microorganisms. The key strength emerged from using multiple assemblers then selecting the optimal result, as different tools perform better with different types of genomes and data qualities.
| Assembler | Average Runtime | Contiguity (N50) | Completeness (BUSCO %) | Best For |
|---|---|---|---|---|
| NextDenovo | Fast | High | High | Standard genomes |
| Flye | Moderate | High | High | Balanced needs |
| Canu | Slow | Moderate | High | Accuracy-critical work |
| NECAT | Fast | High | High | Large datasets |
| Miniasm | Very Fast | Variable | Variable | Draft assemblies |
This comprehensive approach proved particularly valuable for the Candida auris sample, given its clinical significance as an emerging multidrug-resistant pathogen. Complete genome assembly enabled researchers to identify not just individual resistance genes, but their genomic context—information crucial for understanding how resistance develops and spreads.
Successful microbial genomics relies on specialized reagents and tools at each experimental stage:
| Category | Specific Examples | Function |
|---|---|---|
| DNA Extraction Kits | Circulomics Nanobind, QIAGEN Genomic-tip | Obtain high-molecular-weight DNA without shearing |
| Library Preparation | ONT Ligation Kits, PacBio SMRTbell | Prepare DNA for sequencing with appropriate adapters |
| Sequencing Kits | ONT Flow Cells, PacBio SMRT Cells | Generate long-read sequence data |
| Bioinformatics Tools | Prokka, BRAKER3, InterProScan | Analyze sequence data and extract biological insights |
| Reference Databases | KEGG, eggNOG, CAZy | Annotate gene functions and metabolic pathways |
Quality DNA extraction is particularly crucial, as the platform developers note: "The extraction must be pure and of high molecular weight. Any damage to the DNA or contamination can result in poor performance, lower read lengths, and even affect the library preparation step" 7 .
Long-read sequencing is expanding our knowledge of microbial diversity at an unprecedented pace. A recent landmark study published in Nature Microbiology used advanced long-read sequencing to recover 15,314 previously undescribed microbial species from soil and sediment samples, expanding the phylogenetic diversity of the prokaryotic tree of life by 8% 3 .
As these technologies become more accessible through platforms like MIRRI-IT's service, we can anticipate accelerated discoveries in areas ranging from human health and disease to environmental conservation and industrial biotechnology. The ability to completely sequence and annotate microbial genomes efficiently will help researchers develop new antibiotics, create sustainable agricultural solutions, and harness microbial capabilities for environmental cleanup.
As one research team optimistically notes, their platform positions itself as "a valuable tool for routine genome analysis and advanced microbial research" 1 —democratizing access to cutting-edge genomic technologies for researchers worldwide.
The development of comprehensive, user-friendly platforms for long-read microbial genome analysis represents more than just a technical advancement—it marks a fundamental shift in how we explore the microbial world. By making sophisticated genomic analyses accessible to researchers without specialized computational expertise, these services are accelerating our understanding of the microorganisms that shape our health, our environment, and our world.
As long-read technologies continue to evolve—becoming more accurate, more affordable, and more accessible—we stand at the threshold of a new era of discovery. The invisible universe of microbes is finally becoming visible, revealing secrets that will help us address some of humanity's most pressing challenges. The genomic revolution in microbiology has begun, and its potential is limited only by our curiosity to explore it.