When Genomes Go Rogue

The secret to cracking funky genomes may lie in tiny bubbles…

Semi-permeable capsules offer an inventive new way to study genomic instability

Your genome is one huge drama queen; and it has good reason to be. Dozens of delicate processes could go wrong, every time a cell divides: bases could get mispaired, replication forks can stall or collapse [1], sunlight or chemicals may oxidize bases [2], proteins might misfold, and whole chromosomes may not separate during cell division [3]. However, for all these issues, your cells have a thorough emergency response kit.

Proofreading enzymes and mismatch repair watch out for mistakes during replication [4]. Specialized enzymes help reverse chemical damage [5]. Homologous recombination and non-homologous end joining stick together broken strands. Transcription-coupled repair and RNA surveillance keep an eye out for transcript errors. These are just a portion of your cells genomic arsenal.

The sum of all of these risks is referred to as genome instability, the ‘range of DNA alterations… which irreversibly change the information content of the genome’ [6]. For most organisms that’s a big problem, possibly contributing to aging and cancer [6][7]. But for some, especially pathogens, a bit of instability is a survival hack: more variation can mean faster adaptation.

“While genomic integrity is typically vital for cellular function, moderate levels of instability… can facilitate survival under environmental stress by quickly diversifying phenotypes in a cell population”.

Today’s preprint comes from a team of 4 researchers from the Experimental and Molecular Parasitlogy Units of the Institute of Tropical Medicine Antwerp in Antwerp, Belgium.

http://biorxiv.org/lookup/doi/10.1101/2025.09.10.675331

(Side note: these preprints keep introducing me to some of the coolest research niches out there, I would love to be a molecular parasitologist!) They sought to combat one of the biggest obstacles in genome instability research; low resolution.

🔬 Imagine a really blurry pair of glasses…

Most cell sequencing methods lump thousands if not millions of cells together to produce expansive datasets that present an “average” genome. ‘Quirky’ alleles, introduced by genomic instability, that may drive adaptation or survival under stress are drowned out in the crowd.

**P.S: ‘Quirky alleles’ is not scientific language

This bulk sequencing is a huge roadblock in studying genomic heterogeniety — cases where different mutations in different genes lead to the same observable outcome, usually a disease or condition.

The obvious fix is single-cell resolution, looking at each genome on its own to see the anomalous quirks. However, compared to single-cell RNA and chromatin methods, single-cell DNA sequencing (scDNAseq) has a long way to go. There are existing methods of scDNAseq however as Negreira et. al. explain “currently available options are often limited in throughput, as cells are usually manually processed in individual PCR plate wells, restricting the number of sequenced cells to the plate’s capacity.”

This issue of throughput is central to efficient cell sequencing methodology, it refers to the amount of DNA that can be ‘read’ at the same time.

This figure from 10x Genomics shows how “single cell RNA-seq reveals cellular heterogeneity that is masked by bulk RNA-seq methods.”

The Amplification Roadblock

One major hurdle in scDNAseq is that a single cell just doesn’t have that much DNA to work with, only a fraction of what is needed for sequencing. To make it readable, researchers have to do molecular photocopying though a process called single-cell whole genome amplification (scWGA).

In practice, scWGA is more like a dodgy office copier [8]. Some regions of DNA are amplified excessively with others being ignored, some fragments can get stitched together creating totally new and artificial sequences; and the worst part is, these copying errors easily appear as genuine mutations.

This error-prone step is the main reason why scDNAseq is so far behind other single-cell sequencing techniques.

So, we have a group of researchers who want to study genomic instability, those weird gene quirks. To do this, they need a sequencing method with single-cell resolution to zoom in on all of the individual cell DNA. The existing techniques used to do this are slow and inefficient. What do they do?

🧪 Tiny bubbles may be the future of DNAseq

The Antwerp team came up with a two-part solution.

Step one: Semi-permeable capsules (SPCs).

First, instead of the impermeable oil droplets used in conventional techniques, the researchers opted for semi-permeable capsules. These are tiny, porous balloons that hold a cell inside but allow for small molecules to flow in and out. This means that can carry out multi-stage genomic reactions like adding enzymes or changing buffers, without having to pack all the ingredients into the capsules from the start.

SPCs are also ridiculously durable, able to survive harsh chemicals used to dissolve cell walls, frigid –80˚C storage temperature, and boiling 100˚C reaction conditions. They’re flexible, scalable to millions of cells per experiment whilst keeping each cell isolated.

Step two: Primary template-directed amplification (PTA).

Rather than the inaccurate scWGA copier, the used PTA as an alternative, which produces more uniform coverage and fewer copy errors. This allowed them to reconstruct the genomes of hundreds of single cells with far more accuracy and even gave them the ability to for a full picture (or karyotype) of the chromosomes of hundreds of individual cells and to detect distinct patterns between them.

Together, SPCs and PTA led to some particularly spectacular outcomes but before we get into that… what on Earth did they sequence?

🦠 Leishmania

This media comes from the Centers for Disease Control and Prevention’s Public Health Image Library (PHIL), with identification number #468.

This protozoan parasite is the culprit behind leishmaniasis, a “tropical disease transmitted by sandflies that is most commonly seen in Europe, Africa, Asia, and Latin America” [9]. It is classed as a neglected tropical disease as it disproportionately affects lower-income communities in developing countries of the Global South [10].

Despite its tragic pathology, from a genomics perspective Leishmania is fascinating. Unlike our strictly regulated human genomes, Leishmania display extraordinary genomic plasticity.

A brief word on ploidy

Most organisms usually have a balanced set of chromosomes (the structural forms of DNA) with the same number of copies for each one, this number of copies is a cells ploidy. Most human cells (with the exception of sex cells called gametes) are diploid meaning they have two sets of chromosomes hence a ploidy of two. This equal number of copies of all chromosomes is called euploidy.

Leishmania, however, almost always has extra copies of chromosome 31, while most of the other chromosomes usually appear in pairs. But this isn’t fixed as the parasite’s chromosomes are highly unstable, and the number of copies is not always the same across all chromosomes (the opposite of euploidy: aneuploidy). This means that even within a single population of Leishmania, different cells can have different chromosome combinations, creating a kind of genetic “mosaic.”

Adaptation Through Instability

Even more fascinating is that these differences weren’t just random static. The Antwerp team found patterns of chromosome copy number that correlated with environmental pressures. Leishmania’s genomic instability allowed it to undergo active adaptation. By reshuffling its genome, the parasite tunes how much RNA and protein it produces, giving it an evolutionary advantage against stressors like drugs or immune defenses. Lets look at the wider results to get a better understanding of this…

A well-known example of aneuploidy is trisomy 21, the genetic difference that causes down syndrome. Notice how there are three chromosome 21s instead of two like the rest of the chromosomes?

📊 The Results

🤝 SPC + PTA for the win!

The team tested several SPC libraries using both the old WGA methods and the alternative PTA method. One of the WGA trials was so unreadable that it got tossed out completely (yikes!); amongst the remaining samples PTA samples gave them way more readable DNA, with deeper coverage per cell.

Even when the team adjusted for read counts, PTA still came out on top. Not only could it analyze more data, it could so it more consistently. The results put PTA very close to what used to be considered gold-standard single-cell analysis platforms.

The one caveat: PTA did show some sensitivity to the exact reagents used, with tiny differences between batches. Still, compared to the unreliable nature of scWGA, PTA is a major upgrade for single-cell DNA sequencing.

🗺️ Single-Cell Karyotyping revealed a mosaic of cell variation

Using SPC + PTA, the researchers were able reconstruct the full karyotype of hundreds of individual Leishmania cells.

They found that the Leishmania genomes showed aneuploid mosaicism (their cells contain a varied mix of chromosome numbers.) Chromosome 31, as expected, almost always exhibited extra copies. But, for the first time, the high resolution allowed the team to see just how varied chromosomes were between cells: some cells were diploid across most chromosomes, others had random copy numbers across a number of different chromosomes.

Importantly, this genetic variation was not just random noise. In some cases, shifts in copy number aligned with environmental stress conditions, such as drug exposure, suggesting that the parasite was not simply randomly shuffling its genome but was actively experimenting with new combinations in the hopes that one might offer an advantage. Genomic instability was being harnessed as a survival mechanism.

💊 Single-Cell Resolution exposed varying levels of drug resistance

Strains of a pathogen with different versions of genes can change up the course of an infection in different ways, and yet standard diagnostic tools almost never pick them up. Bulk sequencing averages everything together, creating an oversimplified picture. When parasites are taken from patients and grown in culture, the resulting genomes often look very different from the original infection; likely because a few strains are expressed by the sequencing while the rest vanish.

This is exactly where single-cell DNA sequencing shines. To test whether their method could separate genomically diverse mixtures, the Antwerp team created artificial infections by mixing two Leishmania donovani strains together. The methodology clearly distinguished the two genotypes at the single-cell level.

The team pushed further by focusing on genes already known to be linked with drug resistance and that’s when they hit the jackpot. One strain of Leishmania showed loads of hidden variation across key genes. Bulk sequencing had completely missed this, but single-cell sequencing revealed it loud and clear.

This pattern suggests heteroresistance, sub-populations within the same infection responding differently to treatment. Earlier studies on miltefosine resistance hinted that Leishmania adapted not by inventing new mutations, but by selecting for pre-existing variants that had been invisible to bulk sequencing but detectable with single-cell approaches.

Conclusion

Evidently, this new SPC + PTA methodology has serious potential, it could finally help scDNAseq catch up to other single-cell sequencing methods. What I find most fascinating about this is the many potential applications for semi-permeable capsules. Though they are not a novel discovery, their use in -omics (branches of biology concerning the studies of sets of biomolecules in organisms; e.g. genomics, proteomics) is relatively recent.

Companies like Atrandi Biosciences are pushing this frontier by making SPC technology more accessible to labs and breaking down the barrier between flashy proof-of-concept and actionable, real-world impacts. And such is the nature of science! When technologies like these escape their suffocating niches and become more broadly deployable, more discoveries can be made and more open science can be conducted.

Thank you for following along, this has been one long post! And I am now officially back from my hiatus, with lots in store!

Footnotes

Hey y’all! I am still trying to gauge what is a good length for a post. I had a lot to say here but in the end I felt as though the pace was dragging. Any feedback and suggestions would be much appreciated!

The methodology used in this study is such a treat. And I say that both as a molecular biology student but also as a science communicator. This is a perfect example of how intuitive and digestible science can be if presented properly.

I absolutely love the semi-permeable capsules. They remind me of microscopic bubble tea pearls. I recommend checking Atrandi Biosciences at their beautifully-designed website for more information: https://atrandi.com/

Their incredible graphics are a great way to visualize the technology behind SPCs:

Illustration showing the structure of an SPC with aqueous core and sieve-like semi-permeable shell “that keep the cells and nucleic acids partitioned inside, while allowing the free flow of the surrounding solution”. — Atrandi Biosciences

Works Referenced

Negreira, G. H., Monsieurs, P., Dujardin, J.-C., & Domagalska, M. A. (2025). A novel high-throughput single-cell DNA sequencing method reveals hidden genomic heterogeneity in the unicellular eukaryote Leishmania. https://doi.org/10.1101/2025.09.10.675331

Single cell RNA-seq: An introductory overview and tools for getting started | 10x Genomics. (n.d.). Retrieved September 21, 2025, from https://www.10xgenomics.com/blog/single-cell-rna-seq-an-introductory-overview-and-tools-for-getting-started

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[2] Kciuk, M., Marciniak, B., Mojzych, M., & Kontek, R. (2020). Focus on uv-induced dna damage and repair — disease relevance and protective strategies. In International Journal of Molecular Sciences (Vol. 21, Issue 19, pp. 1–33). MDPI AG. https://doi.org/10.3390/ijms21197264

[3] Potapova, T., & Gorbsky, G. J. (2017). The consequences of chromosome segregation errors in mitosis and meiosis. In Biology (Vol. 6, Issue 1). MDPI AG. https://doi.org/10.3390/biology6010012

[4] Bulock, C. R., Xing, X., & Shcherbakova, P. v. (2020). Mismatch repair and DNA polymerase δproofreading prevent catastrophic accumulation of leading strand errors in cells expressing a cancer-associated DNA polymerase ϵ variant. Nucleic Acids Research, 48(16), 9124–9134. https://doi.org/10.1093/nar/gkaa633

[5] Chatterjee, N., & Walker, G. C. (2017). Mechanisms of DNA damage, repair, and mutagenesis. In Environmental and Molecular Mutagenesis (Vol. 58, Issue 5, pp. 235–263). John Wiley and Sons Inc. https://doi.org/10.1002/em.22087

[6] Vijg, J., & Montagna, C. (2017). Genome instability and aging: Cause or effect? Translational Medicine of Aging, 1, 5–11. https://doi.org/10.1016/J.TMA.2017.09.003

[7] Swift, L. H., & Golsteyn, R. M. (2016). The Relationship Between Checkpoint Adaptation and Mitotic Catastrophe in Genomic Changes in Cancer Cells. Genome Stability: From Virus to Human Application, 373–389. https://doi.org/10.1016/B978-0-12-803309-8.00022-7

[8] Estévez-Gómez, N., Prieto, T., Guillaumet-Adkins, A., Heyn, H., Prado-López, S., & Posada, D. (2018). 2nd revision — Missassignment problems — Comparison of single-cell whole-genome amplification strategies. BioRxiv. https://doi.org/10.1101/443754

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[10] Okwor, I., & Uzonna, J. (2016). Social and Economic Burden of Human Leishmaniasis. The American Journal of Tropical Medicine and Hygiene, 94(3), 489. https://doi.org/10.4269/AJTMH.15-0408