From corals and camels to companion animals, biologists are increasingly turning to single-cell RNA-seq (scRNA-seq) to explore organisms that fall outside the mouse and human comfort zone. The payoff is vast: novel cell types, comparative evolution, and veterinary biomarkers. The road, however, is anything but plug-and-play. Below, we outline four pain points our team consistently encounters with non-model sequencing requests from our clients, along with the workarounds that have served us well.
Incomplete 3' UTR annotations cripple 3' kits
Most droplet platforms capture only the terminal ~100 bp of each transcript. When those 3′ ends are absent or misplaced in the reference genome, the sequencer dutifully records reads that later fail to align, causing entire genes to disappear from the expression matrix. Filling in missing 3'UTR information can be a whole separate project in itself, requiring specialised tools and often long-read sequencing. Monitoring the proportion of poly‑adenylated yet unmapped reads serves as an early diagnostic for annotation gaps and can prevent costly re‑sequencing.
Immune-repertoire add-ons are species-locked
To profile T- or B-cell repertoires effectively, researchers rely on primers that capture the highly variable complementarity-determining regions. Because commercial enrichment kits target only human and mouse sequences, they fall short for other species. When projects involve species such as dogs, cats, or pigs, teams must design custom constant-region primers. This step typically takes several weeks and requires a dedicated pilot lane to confirm specificity. Consequently, project timelines should account for this extra design and validation phase from the very beginning.
Popular chemistries are not universal
Commonly used chemistries are not one-size-fits-all. Probe-based 10x Genomics Chromium Single Cell Gene Expression Flex shines in human and mouse studies because its stock probe panel recognizes only those transcripts. Custom probe sets are possible, but they require a separate design workflow, which adds time and cost.
If you’re working with draft or incomplete genomes, full-length or random-primed methods are a safer bet. Platforms such as Parse Biosciences' Evercode (with fixation), Scale Biosciences' QuantumScale, Takara Shasta Total RNA-Seq, and VASA-seq read entire gene bodies without depending on pre-made probes. They handle partial assemblies gracefully and, thanks to some of their fixation options, let you lock in sample quality at collection and sequence later.
Table comparing the different sequencing technologies that may be suitable for the sequencing of non-model species.
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Tissue-Specific Dissociation: the Achilles’ heel of single-cell workflows
Each tissue type behaves differently, so you need a tailored dissociation (and often nuclei-isolation) strategy for every project. If you skip this optimization step, you risk shattered cells and unusable libraries. Tough matrices, fatty samples, or mineral-rich tissue ratchet up shear stress, leading to cell damage and noisy gene expression profiles. Single-nucleus RNA-seq can dodge many of these issues, but you give up valuable cytoplasmic and splicing data in the bargain.
Emerging solutions, such as FixNCut, offer a gentler path by briefly crosslinking samples immediately after harvest and then releasing them under chilled conditions. It’s a brilliant idea, but adoption is slow because there’s no one-size-fits-all fixative, and platform compatibility is still being proven. Bottom line: build dissociation and nuclei-prep optimization into your timeline from day one as it is the make-or-break step for any single-cell sequencing workflow.
Implementation roadmap
Single-cell sequencing has become the go-to tool for exploring biology, and it is now applied in nearly every field, from feline immunology to insect development. At Single Cell Discoveries, we receive, on average, one non-model species project every week, and we have already profiled more than 30 tissue types across 40+ different organisms.
Successful sequencing of non-model species hinges on a handful of clear yet carefully executed steps: start with a thorough audit of the reference genome, validate custom V(D)J primers, plan early for fixation-compatible chemistries, and budget cautiously for nucleic-based protocols. When these pieces are in place, single-cell genomics can illuminate the inner workings of virtually any ethically sampled species, delivering actionable insights without detours or surprises.
Ready to put your unique organism under the single-cell lens? Contact us today to uncover the biology of your chosen models.
Figure showing some of the organisms that Single Cell Discoveries has sequenced.
