Biology subjects

Pokorny, L.

Publications and source records attributed to Pokorny, L..

9 recordsLinked to original sources

bioRxiv2026

Enrichment Probe Sets Combining Universal and Lineage-Specific Targets Help Resolve Recalcitrant Lineages

The combination of target capture sequencing (TCS) with low-coverage whole genome sequencing (lcWGS), an approach known as Hyb-Seq, has allowed the integration of natural history collections into the genomics revolution, transforming biodiversity research. To implement Hyb-Seq, a collection of genomic targets, often nuclear orthologs, is needed to design probes for TCS. In flowering plants, the universal Angiosperms353 probe set has been proven resolutive at multiple evolutionary scales, with caveats. Malpighiales is known to be one of the most challenging flowering plant orders to resolve. Within this order, the clusioid clade ([~]2.2K species, 94 genera, five families) is no exception. To resolve phylogenetic relationships in this recalcitrant clade, we design a custom probe set, the Clusioids626 kit, composed of 39,936 120-mer probes targeting 626 nuclear orthologs ([~]6.6M nucleotides). This probe set includes all Angiosperms353 targets and 273 clusioid-specific ones, carefully chosen taking copy-number, length evenness, and phylo-informativeness into account. We test our probe set on 70 accessions representing all families and tribes in the clusioid clade. On average, 50.4% of TCS reads mapped to our targets, recovering a median of [~]600 orthologs. Relationships for all clusioid families are fully resolved for our nuclear targets. Additionally, 105 plastid coding DNA sequences were retrieved from the lcWGS fraction. A strong cyto-nuclear conflict was detected. The Clusioids626 kit performs better than the universal Angiosperms353 enrichment panel alone. Our kit design workflow can be extended into other lineages for which a universal probe set exists but more resolution is needed.

evolutionary biology↗

bioRxiv2026

Stress-responsive Mycobacterium tuberculosis subpopulations manipulate macrophage polarization and can be targeted to limit inflammation

Tuberculosis is characterized by broad clinical heterogeneity that hinders infection control, with differences in lesion development, progression, and treatment outcomes. This complexity is likely associated with Mycobacterium tuberculosis inherent phenotypic variation and its capacity to diversify under host microenvironmental and antimicrobial stressors. Here, we analyze M. tuberculosis at the single-cell and subpopulation level using fluorescent reporters, imaging, transcriptomic, and functional assays. We identify RNA signatures specific to stress-responsive bacilli with translational potential. Focusing on the clinically validated chaperone GroEL2, we find that it correlates with M. tuberculosis growth rate and stress tolerance in vitro and intracellularly. Furthermore, GroEL2 phenotypic diversity influences innate responses in macrophages, which experience different polarization, in turn affecting GroEL2 expression. We also show that targeting GroEL2 impairs pathogen survival and dampens inflammation. This study provides a link between pathogen phenotypic variation and macrophage fates, with implications for early infection outcomes, local disease progression, and subpopulation-targeted interventions.

microbiology↗

bioRxiv2026

Automated Optimization of Bacterial Tracking Pipelines with TrackMate 8

Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, Microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacteria morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacteria images in TrackMate, (2) a TrackMate-Helper extension for parameter optimization, and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging.

microbiology↗

bioRxiv2025

Unveiling the hidden biodiversity of microbial chemoreceptor genes in the rare biosphere

Chemoreception plays a central role in microbial adaptability, influencing both community structure and interactions with the environment. However, many chemosensitive microorganisms occur at low abundances in natural ecosystems, which has limited their detection and study using conventional metagenomic sequencing. Here, we employed a custom-designed target capture sequencing approach--encompassing all known chemoreceptor genes from both cultured and uncultured microorganisms--to uncover and characterize the vast chemosensory potential and biodiversity within the rarest fractions of the microbiome. Compared to standard environmental sequencing methods, our approach enhanced the detection of chemoreceptor (CR) genes and their associated sensing domains by orders of magnitude across diverse environments, including the rhizosphere, phyllosphere, soil, aquatic ecosystems, the human gut, and bovine rumen. This enabled the identification of thousands of low-abundance chemosensitive microorganisms that remained undetectable using conventional sequencing approaches, including known plant pathogens and symbionts. Phylogenetic analysis of the most divergent CR genes revealed evidence for novel chemosensitive species, potentially representing new bacterial phyla and classes. Our study provides a new perspective on the chemoreception capabilities of environmental microbes and opens new avenues for discovering and characterizing novel microbial sensing mechanisms.

microbiology↗

bioRxiv2024

Integrating target capture with whole genome sequencing of recent and natural history collections to explain the phylogeography of wild-growing and cultivated Cannabis

O_LICannabis has provided important and versatile services to humans for millennia. Domestication and subsequent dispersal have resulted in various landraces and cultivars. Unravelling the phylogeography of this genus poses considerable challenges due to its complex history. C_LIO_LIWe relied on a Hyb-Seq approach (combining target capture with shotgun sequencing), with the universal Angiosperms353 enrichment panel, to explore the genetic structure of wild-growing accessions and cultivars by implementing phylogenomic and population genomic workflows on the same Hyb-Seq data. C_LIO_LIOur findings support the treatment of Cannabis as a monotypic genus (C. sativa L.), structured into three main genetic groups--E Asia, Paleotropis, and Boreal--with clear phylogeographic signal despite significant levels of admixture. The E Asia group was sister to the Paleotropis and the Boreal groups. Individuals within the Paleotropis group could be further structured into three subgroups: Iranian Plateau, C & S China and Himalayas, and Indoafrica. Individuals from the Boreal group split into two subgroups: Eurosiberia and W Mongolia and Caucasus and Mediterranean. Hemp and drug-type landraces and cultivars consistently matched their putative geographic origin. C_LIO_LIThese findings enhance our understanding of the genetic patterns in Cannabis and provide a framework for future research into its current and past genetic diversity. C_LI

genomics↗

bioRxiv2023

Dynamic microfluidic single-cell screening identifies pheno-tuning compounds to potentiate tuberculosis therapy

Drug-recalcitrant infections are a leading global-health concern. Bacterial cells benefit from phenotypic variation, which can suggest effective anti-microbial strategies. However, probing phenotypic variation entails spatiotemporal analysis of individual cells that is technically challenging, and hard to integrate into drug discovery. To address this, we developed a flow-controlled multi-condition microfluidic platform suitable for imaging two-dimensional growth of bacterial cells, compressed inside separate microchambers by a soft hydro-pneumatic membrane. With this platform, we implemented a dynamic single-cell screening for compounds that induce a phenotypic change while decreasing cell-to-cell variation, aiming to undermine the bacterial population, making it more vulnerable to other drugs. We first applied this strategy to mycobacteria, as tuberculosis poses a major public-health threat. Our top hit impairs Mycobacterium tuberculosis via a peculiar mode of action and enhances other anti-tubercular drugs. This work proves that pheno-tuning compounds represent a successful approach to tackle pathogens that are increasingly difficult to treat.

microbiology↗

bioRxiv2022

The Vaccinia virus chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) - the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.

microbiology↗

bioRxiv2021

A Comprehensive Phylogenomic Platform for Exploring the Angiosperm Tree of Life

The tree of life is the fundamental biological roadmap for navigating the evolution and properties of life on Earth, and yet remains largely unknown. Even angiosperms (flowering plants) are fraught with data gaps, despite their critical role in sustaining terrestrial life. Today, high-throughput sequencing promises to significantly deepen our understanding of evolutionary relationships. Here, we describe a comprehensive phylogenomic platform for exploring the angiosperm tree of life, comprising a set of open tools and data based on the 353 nuclear genes targeted by the universal Angiosperms353 sequence capture probes. This paper (i) documents our methods, (ii) describes our first data release and (iii) presents a novel open data portal, the Kew Tree of Life Explorer (https://treeoflife.kew.org). We aim to generate novel target sequence capture data for all genera of flowering plants, exploiting natural history collections such as herbarium specimens, and augment it with mined public data. Our first data release, described here, is the most extensive nuclear phylogenomic dataset for angiosperms to date, comprising 3,099 samples validated by DNA barcode and phylogenetic tests, representing all 64 orders, 404 families (96%) and 2,333 genera (17%). Using the multi-species coalescent, we inferred a "first pass" angiosperm tree of life from the data, which totalled 824,878 sequences, 489,086,049 base pairs, and 532,260 alignment columns. The tree is strongly supported and highly congruent with existing taxonomy, while challenging numerous hypothesized relationships among orders and placing many genera for the first time. The validated dataset, species tree and all intermediates are openly accessible via the Kew Tree of Life Explorer. This major milestone towards a complete tree of life for all flowering plant species opens doors to a highly integrated future for angiosperm phylogenomics through the systematic sequencing of standardised nuclear markers. Our approach has the potential to serve as a much-needed bridge between the growing movement to sequence the genomes of all life on Earth and the vast phylogenomic potential of the worlds natural history collections.

evolutionary biology↗

bioRxiv2018

A Universal Probe Set for Targeted Sequencing of 353 Nuclear Genes from Any Flowering Plant Designed Using k-medoids Clustering

Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost associated with developing targeted sequencing approaches is preliminary data needed for identifying orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm lineage. To maximize the phylogenetic potential of the probes while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, five to 15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order lineages of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order lineages, including all angiosperms.

evolutionary biology↗