Researchers have successfully completed the first on-site, direct whole-genome sequencing of subaerial biofilms (SABs) on a UNESCO World Heritage Site stone monument.
In a pioneering study, Fabiana Soares and co-authors from the University of Coimbra and Genoinseq, Portugal, have developed a portable workflow for direct whole-genome sequencing using Bento Lab and Oxford Nanopore Technology’s MinION device.
Their objectives were to be able to a) identify microbial communities involved in the deterioration of historic stone structures, b) assess their functional traits, and c) flag potential resistances to conservation treatments, conducting all work entirely on-site and outside of a conventional laboratory.
“We present the first on-site direct Whole Genome Sequencing analysis of SABs thriving in a UNESCO World Heritage Site stone monument […] using a combination of the Bento Lab Pro suite and the Oxford Nanopore® MinION™ sequencer.” — Soares et al. (2025)
Investigating The Microbiomes of Coimbra’s Roman Cryptoporticus
As a test case, the team focused on Coimbra’s Roman Cryptoporticus—an approximately 2,000-year-old subterranean Roman gallery located beneath the Machado de Castro National Museum.
Roman engineers built the Cryptoporticus in the 1st Century AD to support the Roman Forum (the city’s center). In the 12th Century, a Bishop’s palace was constructed on top, and today this building houses the Machado de Castro National Museum. In 2019, UNESCO formally included the Museum and the Cryptoporticus in Coimbra’s UNESCO World Heritage designation.
While the overlying building has protected the Cryptoporticus structure from destruction, microbial activity could damage the stonework over time, most notably biofilms living on the stone surfaces that produce acids or degrade minerals by other mechanisms. This made the Cryptoporticus an ideal candidate for investigation using their new portable workflow.
“Microbial proliferation and the action of environmental microbiomes in subaerial biofilms (SABs) can contribute to cultural heritage stone biodeterioration, posing serious challenges for monument conservation.” — Soares et al. (2025)
Setting up a portable whole-genome sequencing laboratory
To trial their mobile workflow, the team set up a temporary laboratory on a bench in a spare room of the museum. Their equipment included:
- Bento Lab for DNA extraction, incubation steps, and gel visualisation
- Quantus portable fluorometer for DNA quantification
- Oxford Nanopore Technology’s MinION for whole-genome sequencing
- A high-performance gaming laptop (ASUS TUF Gaming A16) for data processing
- A vortex device
- A mini fridge and a styrofoam box of freezer blocks for cooling
The researchers extracted total genomic DNA using the Nucleospin® Soil Kit, modifying protocols to suit Bento Lab’s centrifuge limit of 8,000 x G by quadrupling centrifugation times and increasing elution time. They quantified DNA yields using the Quantus fluorometer, and assessed the integrity of high molecular weight DNA using Bento Lab’s agarose gel electrophoresis module.
For sequencing, the researchers prepared a library using the Oxford Nanopore Rapid Barcoding Kit 24 V14, assigning four barcodes per sample and pooling the DNA in equimolar amounts. They used Bento Lab’s thermocycler as a heat block during the incubation steps, and then sequenced their library over 24 hours using a MinION Mk1B and Flongle R10.4.1 flow cell.
Since the flow cell remained viable after the run, they also collected and sequenced a fourth sample from a stone surface in a nearby semi-open area, both as a control and to test the feasibility of reloading a library to increase sequencing output.
To analyse the data, the team used the Galaxy web platform and a variety of bioinformatics tools and reference databases to investigate:
- Microbial taxonomic composition and community diversity
- Functionally characterized genes
- Metabolic pathways related to biodegradation
- Resistomes (the total set of genes linked to antimicrobial resistance for each sample)
We were able to reach the proposed objectives (direct WGS analysis) in an overall timeframe of 38 hours […] on site, in a non-laboratorial context, and obtaining a robust characterization of the SABs traits.” — Soares et al. (2025)
The team found that their workflow was effective and relatively rapid, allowing for comprehensive on-site analysis within just 38 hours. Their key findings included:
- Diverse microbial communities: Bacteria generally dominated the samples, while fungi had varying degrees of abundance and diversity. The authors suggested that the differing community composition between samples appeared to be linked to environmental differences such as light, moisture, and mineral substrate. They detected many groups of microorganisms known to be adapted to saline, marine, or extreme stony environments, and to have tolerance to salts and radiation.
- Functional insights: The analysis of genes related to essential microbial functions revealed that each sample had different but overlapping functional processes, and indicated the key processes operating in each biofilm’s ecosystem.
- Biodegradation pathways: The team detected genes for nitrate assimilation, sulfur metabolism, and carbonic anhydrase activity, suggesting the mechanisms contributing to stone biodegradation.
- Antimicrobial resistance: They detected the presence of genes conferring resistance to metals, drugs, and biocides, with genes varying by sample. However, they didn’t detect any plasmids, suggesting that the resistomes were likely localized and had developed in response to their specific environment, rather than being driven by horizontal gene transfer.
“Altogether, the study […] highlights that the microbial communities on these stone monuments are well-adapted to their environment, emphasizing the need for specific and tailored conservation strategies.” — Soares et al. (2025)
Excitingly, the study demonstrates that portable, cost-effective sequencing workflows using Bento Lab and Oxford Nanopore Technology’s MinION can be successfully deployed on-site to inform conservation strategies for culturally significant stone monuments. This approach opens the door for routine, field-based analyses of heritage biodeterioration using rapid sequencing technology.
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