Biology subjects

Fuchs, J.

Publications and source records attributed to Fuchs, J..

At least 19 recordsLinked to original sources

bioRxiv2026

OPN3 mediates retinal-dependent lipofuscin accumulation and its loss sensitizes keratinocytes to blue-light-induced proteomic remodeling

Lipofuscin is a blue-light-absorbing pigment that contributes to oxidative damage. Whether all-trans retinal (atRAL) contributes to its formation in response to light remains unclear. We asked whether blue-light photosensitization of atRAL promotes lipofuscin accumulation in human keratinocytes and whether this depends on Opsin 3 (OPN3). Blue-light excitation of atRAL reduced mitochondrial and lysosomal viability, impaired autophagic flux, and increased lipofuscin. OPN3 knockdown significantly reduced this accumulation. Label-free data-independent acquisition (DIA) proteomics showed that OPN3 acts at three levels. In the dark, OPN3 loss altered proteome networks associated with autophagy, apoptosis, and interferon-related responses. Under blue light, control cells activated a stress-adaptive program spanning inflammatory regulation, lipid metabolism, and mitochondrial function, which atRAL strongly amplified. This molecular signature, including induction of cellular respiration and ATP-production proteins, was largely absent when OPN3 was silenced. Respirometry showed that blue light suppressed oxygen consumption in both lines over the first 24 h, but a faster recovery in OPN3 knockdown cells at 48 and 72 h was observed. Together, these data define three functions of OPN3: maintaining the basal proteome in a blue-light-independent manner, enabling the adaptive blue-light response, and enabling retinal-dependent lipofuscin formation in keratinocytes.

cell biology↗

bioRxiv2026

Identification and functional analysis of αKNL2 genes in cowpea

Although the KINETOCHORE NULL2 (KNL2) protein is an essential inner centromeri c protein that is crucially important for assembly and functioning of kinetochores, our understanding of its organization, dynamics and function of distinct isoforms in the cells of plant species undergoing mitosis/meiosis is far from complete. In this study, we identified and characterized two KNL2.1/KNL2.2 genes in cowpea. GUS reporter constructs and qRT-PCR revealed that the expression profiles of both genes were variable across organs, with the highest expression in leaves and roots. Using an EYFP gene fusion coupled with immunostaining, it was demonstrated that both KNL2 variants colocalized at centromeres in a cell-cycle-dependent manner. The CRISPR/Cas9 technique was used to generate various in-frame deletion and out-of-frame knock-out knl2 mutants. Single- and double-gene knock-out mutants were generated, and the effects of mutations on plant development and seed setting were analyzed. The results are discussed both with respect to the roles of these proteins in kinetochore assembly and in the context of using KNL2 genes for in vivo production of haploids in cowpea. Significance statementThis study identifies two paralogous KNL2 genes in cowpea and reveals their functional redundancy during centromere assembly and essential role in seed development. These findings expand our knowledge of kinetochore dynamics and provide a basis for exploring the evolutionary diversification of centromeric proteins in legumes.

genetics↗

bioRxiv2026

Programmed chromosome elimination correlates with the overexpression of cohesin and additional B chromosome-encoded genes in Aegilops speltoides

Programmed chromosome elimination is a highly controlled developmental process in which specific chromosomes are selectively lost from defined cell types during development. Despite its broad occurrence across plants and animals, the molecular mechanisms driving the tissue-specific elimination of chromosomes remain largely unresolved. Here, we exploit the root-specific elimination of supernumerary B chromosomes in Aegilops speltoides as a tractable model to identify the genetic basis of programmed chromosome loss. A high-quality, chromosome-scale genome assembly was generated, assigning 398 Mb of sequence to the Ae. speltoides B chromosome. Transcriptome profiling across seven tissue types representing chromosome elimination-active, elimination-negative, and B chromosome nondisjunction conditions identified 3,262 genes consistently upregulated in elimination-associated tissues, including 1,035 B genes. Stepwise subtraction of genes expressed in post-elimination and B chromosome-retaining reference tissues, followed by intersection with genes expressed during B nondisjunction in anthers, identified a candidate gene set enriched for chromosome segregation functions. From this set, we prioritized SYN2-B, a B chromosome-encoded cohesin -kleisin subunit whose Arabidopsis thaliana ortholog AtSYN2 induces chromosome bridges and micronucleus formation when misregulated. CENH3-B, an -type centromeric histone variant identified through GO enrichment analysis of B genes expressed in elimination-associated tissues, was shown to be incorporated in centromeres of both A and B chromosomes by transient gene expression assays using protoplasts and 3D structured illumination microscopy. These findings support a model in which B chromosome-encoded perturbations of cohesin activity and centromere composition contribute to selective B chromosome nondisjunction and their elimination in root tissues. Moreover, the SYN2-B promoter is enriched for ethylene response factor-binding sites compared to its A-encoded paralog, suggesting that ethylene is implicated in the root identity pathway driving root-specific B chromosome elimination.

genetics↗

bioRxiv2026

Holocentromere diversity in Cyperaceae: contrasting repeat organisation in Mapanioideae and Cyperoideae

Centromeres ensure accurate chromosome segregation and are typically confined to a single, localised region in monocentric chromosomes. In contrast, holocentric chromosomes exhibit kinetochore activity distributed along the chromosome length. Although holocentricity is widespread in Cyperaceae, the composition and organisation of these centromeres, as well as their evolutionary diversification, remain poorly understood. Here, we investigated centromere organisation in representatives of the subfamilies Mapanioideae (Hypolytrum schraderianum Nees) and Cyperoideae (Cladium mariscus (L.) Pohl) by combining genome assemblies, repeatome characterisation (RepeatExplorer), fluorescence in situ hybridisation (FISH), and immunolocalisation. Comparative synteny analyses incorporating the genomes of Rhynchospora breviuscula (n = 5) and Carex littledalei (n = 29) identified conserved blocks, eventually expanding almost whole chromosomes of H. schraderianum (n = 30) and Cl. mariscus (n = 39), despite divergent chromosome numbers and deep evolutionary distances within Cyperaceae. Mobile elements showed very low abundances and were uniformly dispersed, with Ty1/Copia Angela being the most abundant in both species. In Cl. mariscus, holocentromeres showed an extended distribution of centromere- and kinetochore-associated proteins along the chromosomes, largely colocalised with two satellite DNA repeats that form dispersed clusters. In contrast, H. schraderianum also displayed kinetochore signals along chromatids, but the most abundant satellite DNA family was enriched in distal and interstitial chromosomal regions rather than interspersed along the chromatids. Together, these results reveal different genomic architectures underlying holocentric organisation in phylogenetically distinct Cyperaceae lineages, suggesting that holocentromeres in this family have diversified with variation in centromere organisation in regard to its association with repetitive DNA.

evolutionary biology↗

bioRxiv2026

Bioclimatic variables influence the strength of purifying selection on mitochondrial DNA in an avian clade (Aves: Piciformes)

Mitochondrial loci were for long considered as markers of choice to reconstruct phylogenies. The development of high-throughput sequencing over the past two decades fostered the sequencing of mitogenomes, allowing further macroevolutionary questions to be tested. Several biological traits of birds (e.g. body mass, migration distances) have been related to mitochondrial substitution rates. Environmental parameters in ectothermics vertebrates, and potentially in endotherms, have been further suggested to impact substitution rates for specific taxa or loci. Yet, the relative importance of biological traits versus bioclimatic variables is unknown because the former were not systematically controlled for in studies that underlined the effect of the bioclimatic variables. To assess the importance of bioclimatic variables on selection regimes, we analysed the thirteen mitochondrial protein-coding genes for 176 Piciformes (toucans, honeyguides, woodpeckers), a clade with homogeneous life-history traits that can be found in diverse bioclimatic environments. Our analyses highlighted a negative relationship between temperature annual range and the non synonymous to synonymous substitutions ratio. The higher purifying selection in temperate environments may be a result of the strong constraints on maintaining an optimal metabolism in broader climatic variations. Our results further highlight that care should be taken when applying general mitochondrial clocks to estimate divergence times among avian lineages distributed in different climatic conditions.

evolutionary biology↗

bioRxiv2026

Self-organized hemanoids derived from human iPSCs create a niche that produces definitive extraembryonic hematopoiesis.

Manufacturing red blood cells (RBCs) from human induced pluripotent stem cells (iPSCs) can improve our understanding of embryonic erythropoiesis, foster innovative treatments for RBC-related diseases, and ultimately address clinical blood supply shortages. However, existing systems face low efficiency, enucleation failure, and uncertainty about the develop-mental wave of cultured RBCs. We successfully used self-organized hemanoids to improve iPSC-derived RBC generation. Based on the hypothesis that cellular interactions and 3D organization promote hematopoietic cell fate, we aimed to thoroughly characterize hemanoids. We visualized the spatiotemporal emergence of hematopoiesis by generating a CD43-GFP reporter iPSC line. Imaging and spatial transcriptomics analysis provided de-tailed insight into the hemanoid architecture, identifying stromal cells and hepatoblasts as potential erythropoiesis-supportive elements. The developmental stage mirrors extraembryonic hematopoiesis. Given the difficulties of accessing these early stages in vivo, our system offers a platform not only for further clinical translation but also for exploring hu-man embryonic blood wave dynamics.

developmental biology↗

bioRxiv2025

Extended nuclear glycosylation is a common post-translational modification

In eukaryotes, glycans modify proteins in the secretory pathway and the extracellular space. Aside from nucleocytoplasmic O-GlcNAc, glycosylation is not considered a relevant post-translational modification in other cellular compartments. Here, we challenge this long-standing paradigm by showing that extended O-glycans are commonly found on intranuclear proteins. Through comprehensive genetic and biochemical analyses, we conclusively demonstrate that these O-glycans stem from the secretory pathway, yet are found on nuclear proteins across mammalian cell lines and primary cells. Using knock-out cell lines, we show mechanistically that nuclear glycans are shuttled to the nucleus via active vesicular transport. We identify several of these intranuclear glycoproteins as RNA-binding proteins, including KHSRP/FUBP2, RBM12, and RPP30. Lastly, we show that site-specific glycosylation of RPP30 is crucial for effective tRNA processing. Overall, our findings suggest a much broader role for glycosylation in regulating cellular functions and open up investigation into the role of glycans in more biological processes.

molecular biology↗

bioRxiv2025

Interactions of the LINE-1 encoded ORF1p with proteins and chromatin converge on a role in neuronal physiology

Retrotransposons are emerging as novel regulators of embryonic and brain development. We recently demonstrated that the LINE-1-encoded protein ORF1p is abundantly expressed in adult mouse and human neurons, although its function remains unclear. Here, we characterize the ORF1p interactome in differentiated mouse and human neurons using mass spectrometry and identify novel partners implicated in gene regulation and neuron-specific processes. ORF1p localizes not only to neuronal nuclei, where it associates with chromatin under steady-state conditions, but also to neurites, supporting a role in neuronal physiology. To further explore its nuclear functions, we sorted human post-mortem neurons with high or low nuclear ORF1p levels and performed ORF1p knockdown in cultured human neurons, followed by chromatin accessibility assays. Both approaches revealed consistent patterns of differential chromatin accessibility dependent on ORF1p. Loss of ORF1p also led to the downregulation of long, neuron-specific genes and altered neurite morphology. Together, these findings point to a physiological role of ORF1p in post-mitotic neurons, mediated through converging interactions with proteins and chromatin.

neuroscience↗

bioRxiv2025

Hybridity of mainly asexually propagating duckweeds in genus Lemna - dead end or breakthrough?

O_LIThe cosmopolitan, mainly vegetatively propagating, organ-reduced monocotyledonous aquatic duckweeds are the smallest and fastest growing angiosperms, distributed world-wide and flower rarely in nature. Recently, we reported intra- and interspecific hybrids and ploidy variants in the genus Lemna. Thus, contrary to the expectation, sexual propagation may occasionally occur within and between Lemna species. Our main goal was to uncover whether the ecologically successful hybrids are evolutionary dead ends, or initiate further speciation and novel sexual recombination. C_LIO_LIWe investigated flower development, pollen viability, seed set and seed germination in hybrids and their parental species and characterized genome size and genetic markers in the progenies. C_LIO_LIIntraspecific crosses yielded fertile progeny, but all dihaploid and triploid interspecific hybrids were male sterile. Only an established allotetraploid hybrid reproduced sexually, while colchicine-induced allotetraploids from dihaploids did not re-gain sexual competence so far. C_LIO_LIWe concluded that only established allotetraploid hybrids represent an evolutionary break-through in duckweeds. Our results regarding sexual traits within the duckweed genus Lemna, and the sexual competence of diverse hybrids i) pave the way for further investigation in this understudied field, ii) provide fundamental data regarding the evolutionary potential of duckweed hybrids and iii) are important for future breeding efforts on this emerging crop. C_LI

evolutionary biology↗

bioRxiv2025

Shedding light on the embryo:endosperm balance diversity that pseudogamy can achieve in the polyploid apomictic Psidium cattleyanum (Myrtaceae, Myrteae)

Apomixis is defined as the asexual mode of reproduction through seeds. Nearly all gametophytic apomictic species are polyploid. Psidium cattleyanum, a polyploid species with various described ploidy levels, exhibits apomixis and requires effective pollination for fruiting (pseudogamy). The aim of this study was to determine the frequency of apomixis and sexuality in P. cattleyanum among heptaploid and octoploid accessions, and to analyze the maternal and paternal contribution to endosperm formation. Flow cytometry was employed on mature seeds obtained from hand-pollinated crosses between two octoploid and two heptaploid accessions to determine the ploidy levels of embryo and endosperm. Within the 492 analyzed seeds the ploidy balance of embryo:endosperm ranged from the expected 2:5 (64.2%, pseudogamous apomixis) and 2:3 (4.7%, sexuality involving reduced gametophytes), to unexpected values, including 1:3 (0.4%),), 2:6 (2.4%), 2:7 (0.2%), 3:5 (8.8%), 2:3:5 (18.5%) and 2:4:6 (0.8%), associated with polyhaploid parthenogenesis, pseudogamous apomixis involving reduced and unreduced gametophytes, and female gametophytes with two or three polar nuclei in the central cell. The interpretations and implications of these results for apomictic polyploid species and their consequences for wild populations are discussed.

genetics↗

bioRxiv2025

Kinetochore mutations and histone phosphorylation pattern changes preceded holo- and macro-monocentromere evolution

Centromeres are essential for kinetochore assembly and spindle attachment. While chromosomes of most species are monocentric with a single centromere, a minority exhibit holocentricity, with a centromere along the chromatid length. Sporadic emergence of holocentricity suggests multiple independent transitions. To explore this, we compared the centromere and (epi)genome organization of two sister genera with contrasting centromere types: Chamaelirium luteum with large "macro-monocentromeres" and Chionographis japonica with holocentromeres. Both exhibit chromosome-wide histone phosphorylation patterns distinct from typical monocentric species. Kinetochore analysis revealed similar chimeric Borealin in both species, with additional KNL2 loss and NSL1 chimerism in Cha. luteum. The broad-scale synteny between both genomes supports de novo holocentromere formation in Chi. japonica. Despite sharing features with both centromere types, macro-monocentromeres do not represent a direct link between mono- and holocentromeres. We propose a model for the divergent evolution involving kinetochore gene mutations, altered histone phosphorylation patterns, and centromeric satellite DNA amplification.

evolutionary biology↗

bioRxiv2025

Triploidy is prominent in the duckweed Lemna minor complex

Duckweeds (Lemnaceae Martinov) are aquatic monocotyledonous flowering plants comprising five genera and 35 recognized species, known for being the smallest and fastest-growing flowering plants on Earth. Many species are morphologically indistinguishable due to their highly reduced structures, yet molecular evidence suggests that visually similar clones may represent distinct species or hybrids. For example, clonal accessions of the globally distributed Lemna minor in the Landolt Duckweed Collection exhibit genome size variations of several hundred megabases (Mb), raising questions about their taxonomic classification and evolutionary origins. We analyzed 58 presumed L. minor clones to resolve these relationships using a comprehensive suite of methods, including whole-genome sequencing (WGS), flow-cytometric genome size measurements, molecular markers, chromosome counting, and genomic in situ hybridization (GISH). Our findings reveal extensive genome plasticity within the "Lemna minor complex," identifying diploid and triploid L. minor clones, as well as di-haploid and triploid interspecific hybrids called L. x japonica (L. minor x L. turionifera), L. x mediterranea (L. minor x L. gibba), and a novel African-clade distinct from known L. minor lineages. Triploidy was prevalent, occurring in 29% of the clones, and was associated with enhanced growth under optimal conditions but reduced performance under high light and temperature. These findings highlight the widespread role of triploidy, cryptic species, and hybridization in the L. minor complex, emphasizing the importance of multiple approaches for accurately classifying duckweed species and understanding their evolutionary trajectories.

plant biology↗

bioRxiv2025

CRISPR-CISH: An in situ chromogenic DNA repeat detection system for research and life science education

In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3 biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.

genetics↗

bioRxiv2025

R-Spondin1 regulates fate of enteric neural progenitors via differential LGR4/5/6-expression in mice and humans

Regeneration and cytodifferentiation of various adult epithelial stem cell compartments are controlled by the Wnt-agonist R-Spondin1 (RSPO1) and the Leucin-rich-repeat-containing G-protein-coupled-receptors (LGR4/5/6). We hypothesized that RSPO1-LGR-signaling also is involved in regulating neuro-regeneration and homeostasis of the postnatal enteric nervous system (ENS). If applied to murine and human ENS-progenitors, RSPO1 led to an increased proliferation, followed by enhanced enteric neurogenesis. This coincided with an upregulation of LGR4 expression during ENS-progenitor proliferation in vitro. In contrast, we observed a reduced proliferation in ENS-progenitors expressing LGR5, while LGR6 was not expressed by proliferative ENS-progenitors. Instead, LGR5 and LGR6 expression increased over the course of induced neuronal differentiation, consistent with the in vivo expression. LGR-receptor expression therefore might represent a previously unknown mechanism influencing the fate decision of ENS-progenitor cells between proliferation and neuronal differentiation. Thus, our study is essential for our understanding of regenerative aspects of the postnatal ENS in health and disease.

developmental biology↗

bioRxiv2025

To hybridise or not to hybridise? Systematic review and meta-analysis of woodpeckers

Hybridisation is a common phenomenon among birds in general. Woodpeckers (Picidae) are no exception, as approximately 20% of species are known to hybridise and for many others interspecific mating is suspected. However, the mechanisms and consequences (phenotypic and genetic) of hybridisation are known for only a fraction of woodpecker species. Here, we conduct a systematic review on the literature that deals with hybridisation in woodpeckers and use a meta-analytical approach to examine the available geographical and genetic data. According to available published data, the majority of woodpeckers that hybridise inhabit the Neotropics, followed by the Nearctic and the Palearctic. Hybridisation appears to be less common in the Afrotropic and Oriental regions. As expected, genetic distances are substantially lower between hybridising species pairs than between non hybridising congenerics. This implies that hybridisation is typical for "young" (sister) pairs of species, that typically have similarities in their respective reproductive biology, ecology and ethology, and thus had less time for genetic incompatibilities to build up. These similarities also explain the difficulties experienced in studies on hybrid woodpeckers, but these could be overcome by the use of modern techniques (remote sensing and/or citizen science combining with AI identification and genomic identification). As hybridisation plays a role in woodpecker evolution and ecology it should be considered when conservation policies for rare species are drafted.

evolutionary biology↗

bioRxiv2025

Genome diversity and phylogeny of the section Alatae of genus Lemna (Lemnaceae), comprising the presumed species Lemna aequinoctialis, Le. perpusilla and Le. aoukikusa

O_LIThe Section Alatae of genus Lemna of the monocotyledonous aquatic duckweed family (Lemnaceae) consists of rather diverse accessions with unknown phylogeny and unclear taxonomic assignment. In contrast to other duckweeds, some Alatae accessions, in addition to mainly vegetative propagation, produce readily flowers and viable seeds. We analyzed the genomic diversity and phylogenetic relationship of 52 Alatae accessions. C_LIO_LIFor this purpose, we applied multiple molecular and cytogenetic approaches, including plastid and nuclear sequence polymorphisms, chromosome counting, genome size determination and genomic in situ hybridization in combination with geographic distribution. C_LIO_LIWe uncovered ploidy variation, recurrent hybridization, and backcrosses between diploid species and their hybrids. The latter successfully spread over three continents. C_LIO_LIThe results elucidate the evolution of Alatae accessions (summarized in Fig. 7) and explain the difficult taxonomic assignment of distinct accessions. Our study might be an example for analogous studies to resolve the hitherto unclear relationships among accessions of the duckweed genera Wolffiella and Wolffia. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/632899v1_fig7.gif" ALT="Figure 7"> View larger version (23K): org.highwire.dtl.DTLVardef@d05f08org.highwire.dtl.DTLVardef@f33c5org.highwire.dtl.DTLVardef@ff7adorg.highwire.dtl.DTLVardef@4da6e4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 7C_FLOATNO Hypothetical scenario of evolution within Lemna Section Alatae. In bold are accessions from America. The classically defined diploid species Le. aequinoctialis and Le. perpusilla sensu stricto were confirmed by the applied molecular methods. The previously named Le. aoukikusa accessions were uncovered as tetraploid interspecific hybrids between Le. aequinoctialis and Le. perpusilla. In addition, further tri- and tetraploid hybrids as well as tri- and tetraploid "Backcross hybrids" were detected from the USA and China, respectively. C_FIG

evolutionary biology↗

bioRxiv2025

Comprehensive analysis of 37 composts: microbial indicators for soilborne disease suppression in three plant-pathogen systems

Compost is a valuable amendment for soil and potting substrate when it comes to sup-pressing soilborne pathogens. However, the effectiveness of different composts varies and can not yet be predicted. Microbial communities in compost play a key role in disease suppression, and therefore their composition or specific taxa may serve as indicators of suppressive composts. In this study, we investigated 37 composts from seven commercial compost producers to analyze the association of their bacterial and fungal communi-ties with suppressive activity in three plant-pathogen systems: cress-Globisporangium ultimum, cucumber-Globisporangium ultimum and cucumber-Rhizoctonia solani. Our results underscore that compost suppressiveness is primarily pathogen-specific and, to a lesser extent, host-plant-specific. Suppressiveness was not correlated with physico-chemical properties, microbial activity, or the alpha-and beta-diversity of composts bac-terial and fungal communities. Instead, microbial composition was largely shaped by producer-specific composting conditions and maturation processes, which were not nec-essarily linked to suppressive activity. A more nuanced comparison between the most and least suppressive composts revealed bacterial and few fungal taxa as potential indicators of suppressiveness for each plant-pathogen system. Notably, for G. ultimum-suppression, bacteria from the genera Luteimonas, Sphingopyxis, and Algoriphagus and for R. solani bacteria belonging to the phylum Actinomycetota emerged as promising candidates. ImportanceSoilborne diseases are a major yield-limiting factor in agricultural crop production world-wide, particularly in seedling cultivation. Their control remains a significant challenge and still largely relies on chemical fumigation of soils and steam sterilization of pot-ting substrates. While chemical fumigants are increasingly criticized for their negative environmental impact, sterilization practices in general disrupt beneficial microbial com-munities, making substrates more susceptible to pathogen (re)-infestation. Amending soil or potting substrate with disease-suppressive compost offers a promising alternative. However, the targeted use of compost for plant protection is hindered by variable effec-tiveness and the lack of reliable tools to identify effective composts. This study provides a comprehensive abiotic and biotic characterization of compost, enabling a detailed anal-ysis of the properties associated with suppressiveness. The identification of bacterial and fungal taxa indicative of disease-suppressive composts lays the groundwork for targeted isolation of microorganisms and functional studies, with the ultimate aim of predicting and optimizing compost-mediated disease suppression.

molecular biology↗

bioRxiv2024

varVAMP: automated pan-specific primer design for tiled full genome sequencing and qPCR of highly diverse viral pathogens.

Time- and cost-saving surveillance of viral pathogens is achieved by tiled sequencing in which a viral genome is amplified in overlapping PCR amplicons and qPCR. However, designing pan-specific primers for viral pathogens that have high genomic variability represents a major challenge. Here, we present a bioinformatics command-line tool, called varVAMP (variable virus amplicons). It relies on multiple sequence alignments of highly variable virus sequences and enables automatic pan-specific primer design for qPCR or tiled amplicon whole genome sequencing. The varVAMP software guarantees pan-specificity by two means: it designs primers in regions with minimal variability and introduces degenerate nucleotides into primer sequences to compensate for common sequence variations. We demonstrate varVAMPs utility by designing and evaluating novel pan-specific primer schemes suitable for sequencing the genomes of SARS-CoV-2, Hepatitis E virus, rat Hepatitis E virus, Hepatitis A virus, Borna-disease-virus-1, and Poliovirus. Moreover, we established highly sensitive and specific Poliovirus qPCR assays that could potentially simplify current Poliovirus surveillance. Importantly, wet-lab and bioinformatic techniques established for SARS-CoV-2 tiled amplicon sequencing were readily transferable to these new primer schemes and will allow sequencing laboratories to extend their established methodology to other human pathogens.

bioinformatics↗