International researchers develop practical recommendations to strengthen cancer microbiome research

Published: 25 Mar 2026

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An international team led by researchers from NUS Medicine and A*STAR Genome Institute of Singapore (A*STAR GIS), has published a new paper in Nature Cancer highlighting the importance of greater care, consistency, and rigour in studies reporting microbes in human tumours.

In recent years, advances in DNA sequencing have enabled researchers to detect traces of bacterial, fungal, and viral genetic material in diverse samples. Some studies have reported microbes even in sites traditionally considered microbe-free, such as the brain or the placenta, and findings on microbes in tumours from diverse organs have generated interest in how tumour-associated microbes could influence cancer growth, spread, or response to treatment.

However, the researchers caution that interpreting such signals can be complex. Tumour tissues from diverse organs typically contain extremely low amounts of microbial material or none, making them especially vulnerable to contamination introduced during surgery, sample handling, storage, laboratory processing, or sequencing. Limitations in data analysis and reference databases can further complicate interpretation, leading to microbial signals that may not reflect true biological presence.

“Finding microbial DNA in tumours is like finding a needle in a haystack,” said Associate Professor Niranjan Nagarajan, Department of Biochemistry and Infectious Diseases TRP, NUS Medicine, a lead author of this report. “If appropriate checks and additional validation are not built into the analysis, it becomes very difficult to distinguish genuine microbial signals from background noise.” A/Prof Nagarajan is also Associate Director, AI and Compute, and Senior Group Leader, Laboratory of Metagenomic Technologies and Microbial Systems at A*STAR GIS.

The team reviewed current practices across the rapidly growing field of cancer microbiome research and identified recurring methodological challenges, including insufficient use of negative controls, heavy reliance on bioinformatic analysis alone, and limited validation using independent or complementary methods. They drew parallels with early challenges in ancient DNA research, where the adoption of stringent standards helped mature that field into a reliable scientific discipline.

Importantly, the researchers stress that their report does not question the role of cancer-associated microbes in general. Microorganisms are already known to contribute to certain cancers, particularly in tissues exposed to abundant microbes, such as the gastrointestinal tract. Rather, the paper encourages careful evaluation of claims of diverse tumour-resident microbiomes in low-biomass tissues, especially where supporting evidence is limited.

To help the field move forward, the researchers propose a practical checklist of best practices covering the entire research pipeline—from sample collection and handling, to sequencing, data analysis, and biological validation. Here is a quick summary of the team’s proposed best practices:

Prevent contamination during sample collection

Tumour samples should be collected and handled using strict aseptic techniques to minimise the introduction of microbial DNA during surgery or tissue harvesting.
Environmental sampling controls should be collected alongside tumour samples to identify microbes that may originate from the surrounding environment rather than the tissue itself.
Whenever possible, researchers should analyse matched normal tissue samples collected at the same time as the tumour to help distinguish real signals from contamination.

Maintain clean handling and storage procedures

Tissue samples should be handled and stored under controlled laboratory conditions, ideally separated from areas where DNA amplification takes place to reduce contamination risk.
Handling and storage controls, including blank samples or environmental swabs, should be processed and sequenced to detect contamination introduced during these stages.
For formalin-fixed paraffin-embedded (FFPE) tissues, blank paraffin blocks should be processed and sequenced as additional controls.

Use strict laboratory and sequencing controls

Samples should be processed in clean laboratory environments that minimise nucleic acid contamination.
Key findings should ideally be replicated in independent laboratories or validated using different DNA extraction or library preparation kits.
Blank controls and reagent controls should be included in every sequencing run to identify contamination originating from laboratory reagents or sequencing batches.

Ensure sequencing quality and transparency

Sequencing libraries should be quantified before sequencing to help identify potential contamination signals.
Measures such as dual indexing or avoiding multiplexing should be used to reduce sequencing artefacts such as barcode hopping.
Detailed metadata about sample collection, processing centres, and sequencing batches should be reported so potential technical biases can be assessed.

Apply conservative and transparent bioinformatic analysis

Microbial classification should be performed using high-quality reference databases, including complete human genome references, to reduce false microbial assignments.
Researchers should examine the genomic distribution and alignment quality of microbial reads to confirm that detected microbes show biologically plausible patterns.
Computational methods should be used to identify and remove likely contaminant species, especially in low-biomass samples.

Validate findings using independent experimental methods

Sequencing results should be confirmed using targeted laboratory techniques, such as PCR, fluorescence in situ hybridisation (FISH), or immunohistochemistry.
Spatial imaging methods should demonstrate that microbes are actually present within or near tumour cells, rather than appearing at tissue edges where contamination is more likely.
Evidence should also be sought for viable or metabolically active microbes, for example through RNA detection or culture-based methods.

Provide transparent reporting and multiple lines of evidence

Researchers should clearly acknowledge limitations and describe steps taken to detect or prevent contamination.
Strong claims of tumour-resident microbes should rely on multiple independent lines of evidence, rather than a single sequencing signal.

Please refer to their published study for the full checklist and the reasoning behind those items:

https://www.nature.com/articles/s43018-026-01121-6.

These recommendations emphasise preventing contamination, incorporating appropriate controls, validating findings with multiple independent approaches, and being transparent about data limitations. This work reflects an international collaboration involving scientists from Singapore, the United States, the Netherlands, Denmark, and Australia, bringing together expertise in genomics, computational biology, microbiome science, pathology, and immunology.

“When researchers work with very small signals, even minor background noise can influence the outcome,” said Dr Chia Minghao, Senior Scientist at A*STAR GIS, and co-author of the study. “By putting stronger safeguards in place and confirming results through multiple approaches, the scientific community can improve reproducibility and ensure that future discoveries about microbes and cancer rest on solid foundations.”

Microbes associated with tumours could provide important insights into cancer biology and inform future diagnostics or therapies. At the same time, inconclusive or weakly supported findings can slow progress. By encouraging higher and more consistent standards across the field, the researchers hope to strengthen confidence in cancer microbiome research and support future findings that are reliable, reproducible, and clinically relevant.

View the press release here