MB Talks: Oxford Nanopore Conversations in Microbiology

Massive Bioinformatics

03/02/2025

In our webinar titled “Microbiome Analysis of Skin Flora in Behçet’s Syndrome Patients,” we explored in-depth how sequencing studies conducted with Oxford Nanopore Technologies provide critical findings in Behçet’s Syndrome. For those who missed it, we prepared a summary to share essential insights that may contribute to your research in microbiology:

Behçet’s Syndrome (BS) is a chronic, multisystemic vasculitis affecting various organs and all types of blood vessels, with an unknown etiology. It was first described nearly 90 years ago by Turkish dermatologist Hulusi Behçet. Despite this long history, its cause and pathology remain poorly understood. Patients with Behçet’s Syndrome exhibit a phenomenon known as pathergy, characterized by hypersensitivity. Most patients are pathergy-positive, meaning that when a needle punctures the skin, a papule or pustule develops within 48 hours.

The study discussed in the webinar examined the skin flora of 30 pathergy-positive and 30 pathergy-negative patients. The research was conducted as part of Dr. Betül Saraç’s specialization thesis in internal medicine at Cerrahpaşa Medical School, under the supervision of Prof. Dr. Gülen Hatemi. The microbiology-focused experiments were carried out by Prof. Dr. Ayşe Kalkancı and her team at Gazi University.


DNA Isolation

Prof. Dr. Ayşe Kalkancı explained that various methods such as swabbing, scraping, adhesive tape, and biopsies were used to collect skin samples from patients. The study proceeded with swab samples that provided the highest DNA quality. To obtain both bacterial and fungal DNA from the skin, a guanidine buffer was used during the isolation process, yielding a clean DNA isolate.

DNA Sequencing

The DNA samples were delivered to Massive Bioinformatics for sequencing and analysis. Dr. Ibrahim Halil Miraloğlu described the process and emphasized the suitability of Oxford Nanopore Technologies for bacterial and fungal species identification. For bacterial species identification, the 16S rRNA region, which contains hypervariable regions from V1 to V9, is used. Short-read sequencing technologies cover only the V3-V4 region, while Nanopore’s long-read technology can sequence the entire structure. For fungal species, the ITS1 and ITS2 rRNA regions (including the 5.8S structure) are key, particularly ITS1. Nanopore’s ability to sequence these regions in full provides the advantage of species-level resolution, surpassing short-read technologies. The samples were prepared with Nanopore’s library kit and 16S/ITS1 primers, and amplified regions were tagged with barcodes and adaptors before sequencing on the MinION Mk1C device.

Sequencing with Oxford Nanopore Technologies

The principle of Nanopore’s long-read technology involves threading DNA molecules through motor protein-linked nanopores in a flow cell. As each base passes through the pore, it alters the concentration of ions moving across the membrane, generating an electrical signal. This signal is detected and analyzed to determine the corresponding base sequence through a process called basecalling.

Bioinformatics Analysis

The sequencing data in FASTQ format was processed through various workflows to identify which organisms the DNA sequences matched in databases. This analysis determines the type and abundance of microbial species in the sample, enabling alpha/beta diversity, PCA, PCoA, and biomarker analyses.

  • Alpha diversity: Summarizes the structure of a microbial community in terms of richness (number of taxonomic groups), evenness (distribution of group abundance), or both, measured by indices like Shannon, Chao1, and Simpson’s.
  • Beta diversity: Compares structural differences between groups or samples, observed through PCA and PCoA analyses to identify significant separations between parameters.
  • Biomarker analysis (LEfSe): Identifies statistically significant features (e.g., organisms, genes, functions) that differentiate groups and evaluates their biological relevance.

Dr. Ibrahim Halil Miraloğlu commented on the bioinformatics analysis results: “No significant differences in alpha diversity were detected in the bacterial population. However, beta diversity analyses revealed that the bacterial composition at the genus and species level differed between pathergy-positive and negative groups. For fungi, alpha diversity showed higher species richness in pathergy-positive patients, while beta diversity showed no significant differences between groups. Dominant bacterial and fungal species were also reported.” Prof. Dr. Ayşe Kalkancı highlighted that Behçet’s patients had a dominant Candida population compared to healthy individuals, with Candida auris, a recently discovered species, showing potential as a biomarker for further research.

Q&A Session

Question: What makes Oxford Nanopore’s technology stand out for species identification?

Dr. Ibrahim Halil Miraloğlu: “Even analyzing all nine hypervariable regions of the 16S rRNA may not be enough to distinguish closely related species. For example, distinguishing between E. coli and Shigella can be challenging due to their similar hypervariable regions. Longer reads provide more information, making it easier to identify species and subspecies.”

Question: What are the differences between qPCR and next-generation sequencing in microbiology?

Dr. Ibrahim Halil Miraloğlu: “qPCR uses primers that are available in databases, meaning only predefined species can be detected. This limits qPCR to targeted analyses, preventing unbiased identification of all species present. Next-generation sequencing, however, employs a pathogen-agnostic approach, allowing for comprehensive species discovery.”

Question: What differences did you observe in bacterial composition between pathergy-positive and negative patients?

Prof. Dr. Ayşe Kalkancı: “Unlike fungal composition, we observed no differences in richness within the samples. However, in contrast to the gram-positive dominated composition of healthy individuals, the samples from Behçet’s patients showed a predominance of Proteobacteria. This significant phylum-level shift provided valuable data. Species-level distinctions required genomic analysis, as they could not be identified through traditional phenotypic methods.”

Question: Did you encounter any unusual findings during the bioinformatics analysis?

Dr. Ibrahim Halil Miraloğlu: “No, but I must emphasize that steps like sample collection, storage, and DNA isolation can influence downstream bioinformatics analyses. Careful planning of these steps is crucial to ensure high-quality data.”

Question: What advice would you give to young researchers interested in this field?

Prof. Dr. Ayşe Kalkancı: “It is important to thoroughly review the literature and choose original, unexplored topics. Once a topic is selected, formulating a solid hypothesis and following robust technical methods are essential. I also recommend asking new questions based on your findings and expanding your research scope.”

Dr. Ibrahim Halil Miraloğlu: “Microbiome research has gained prominence, but bacterial studies often take center stage. Do you think the mycobiome has a comparable impact on human life and should be included in microbiome analyses?”

Prof. Dr. Ayşe Kalkancı: “The mycobiome is a relatively new research area, limited for years by technical challenges. The use of Oxford Nanopore’s long-read technology has finally enabled the comprehensive analysis of fungal taxa using ITS regions.”