Published 5.9.2024
Text: Tiina Sävilammi
Image: Shutterstock
Editing: Viestintätoimisto Jokiranta Oy

 

High-throughput next-generation sequencing methods have facilitated the large-scale accumulation of medical and biological research data over the past decades. The so-called metagenomic methods allow for the precise mapping of species in entire ecological communities across all types of ecosystems.

The mapping of species composition in microbial communities has opened new opportunities to observe the interactions of the microbes, both with each other and with the environment and host organisms. With a better understanding of microbial diversity and environmental impact, we will be able to draw advantage from the relevant information, for instance, in the planning of medical care and treatments in the future.

Whole-genome sequencing techniques provide tools to identify and distinguish organisms up to species level. The comparison of the genes contained in the metagenomes of different ecological communities also enables the study of functional variation between the communities, such as the prevalence of antibiotic resistance genes or the quantity of bacteria in soil contributing to carbon sequestration. Moreover, it is possible to examine an individual’s microbiome as an indicator of diseases or to assess, for example, the number of specific digestive bacteria in gut microbiota. The research is, however, slowed down by the fact that the large-scale DNA sequencing required by the new methods is currently quite expensive.

 

Towards faster, easier and less expensive sequencing

Numerous DNA sequencing platforms have been developed, which differ slightly from each other in terms of work phases and reaction setups. They still have one problem in common, namely the cumbersome DNA preprocessing before the actual sequencing. The sample must first be prepared in the laboratory in order to generate a DNA sequencing library of molecules for the sequencing platform to read. Typically, the making of a library involves the cutting of whole-genome DNA into fragments of proper length for a particular platform, followed by the adding of platform-compliant DNA adapters to the fragment ends.

Certain viruses use DNA transposition, also known as gene jumping, for spreading. Through this process, viruses add their own genome to the DNA of the host species by means of the gene jumping mechanism. At the University of Turku, a project is in progress in which we are developing a new method based on the transpositional mechanism of bacteriophage Mu. The aim is to make the generation of sequencing libraries easier and faster, while also lowering costs. The method will not only reduce the number of work phases, but also facilitate the making of sequencing libraries for longer read lengths, up to 50,000 nucleotides.

We have optimised the method for application in a case study in which the power of massive sequencing is utilised to study the association of the gut mibrobiota composition and chronic inflammatory bowel disease (IBD) in horses.

 

Tracing equine IBD with the help of massive sequencing

Both human and equine IBD (Inflammatory Bowel Disease) refer to a group of diseases identified in recent years, which appear to become increasingly common in environments affected by humankind. Currently, we do not yet know precisely the number or prevalence of different forms of equine IBD, or which factors affect their pathogenesis.

In horses, the symptoms do not necessarily reflect stomach problems but, instead, may include behavioural issues or reduced performance, which may result in premature retirement. Diagnosing IBD in horses is difficult because an endoscopic examination requires fasting in advance, transportation to the clinic and tranquilisation. There is also a large margin of error since the diagnosis is often based on general symptoms – a major part of the intestinal track cannot be examined for practical reasons. Despite advances in research over the past years, the prognosis for IBD is still only mediocre.

Studies in other mammals have shown that the composition of gut microbiota is a promising biomarker for IBD. The association of gut microbiota composition and IBD was explored in our recent peer-reviewed pilot study at the University of Jyväskylä. For the study, we employed gut microbiota from healthy horses and horses with IBD, and trained a machine learning model to identify individual horses with IBD on the basis of their gut microbiota. The model managed to accurately predict the health status of previously unknown individual horses. Although the amount of samples was limited, the results of this pilot study indicated the potential of gut microbiota profiling for use as an IBD biomarker.

For microbial identification in the pilot study, we used the DNA mitochondrial sub-unit sequencing method, which is an affordable method. However, the shortness of the marker sequence used in this method is an issue as it does not allow for the identification of bacteria to the species or strain levels. Strain-level research would, however, be essential because of the high bacterial diversity. A particular species may include both strains that contribute to the well-being of the host organism and strains that are pathogenic.

In our project, we aim to produce transposition-based sequencing libraries and to demonstrate the efficacy of the new method by sequencing the metagenomes of gut microbiota in their entirety. Our material will also include fungi and DNA viruses. This will enable us to make observations regarding the relative proportions of different microbial groups and the functioning of microbial communities, including the prevalence of antibiotic resistant bacterial strains that occur in horses in Finland.

 

New insights from molecular data

It is relatively easy to determine the species composition of a microbial ecosystem, and it can also be manipulated on the basis of the composition. Thus, new methods based on the determination of the microbial community structure in the gut or other tissues are expected to offer major applications in the future within human medicine as well.

The new technology we are developing will also include the identification of single circular DNA molecules from the metagenome data. This completely new technology will provide us with new molecular-level information about the surrounding world. It might even prove particularly useful for cancer diagnostics since, according to the latest research, various circular structures are found in the DNA of cancer cells.

 

 

 

 

PhD Tiina Sävilammi is currently working for the Department of Biology at the University of Turku. Previously, Sävilammi has worked in several projects related to the genomics of natural populations run by the Universities of Turku and Jyväskylä. Her doctoral dissertation in 2024 concerned the molecular process of adaptation to environmental change in a salmonid fish. With funding granted by the Sakari Alhopuro Foundation, she is currently engaged in research with an aim of streamlining the production of DNA libraries by using the transposition mechanism of viruses. Sävilammi is interested in practical applications of DNA sequencing, such as the potential of equine gut microbiota as an indicator of chronic inflammatory bowel disease.

 

 

 

Literature:

Savilahti, H., Rice, P. A., & Mizuuchi, K. (1995). The phage Mu transpososome core: DNA requirements for assembly and function. EMBO Journal, 19: 4893-4903.

Sävilammi, T., Alakangas, R.-R., Häyrynen, T., & Uusi-Heikkilä, S. (2024). Gut Microbiota Profiling as a Promising Tool to Detect Equine Inflammatory Bowel Disease (IBD). Animals, 14(16), Article 2396.