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Short-read genome skimming enables molecular barcoding of old myxomycete collections.

This study evaluates the effectiveness of Illumina-based genome skimming for barcoding myxomycete herbarium collections ranging from 29 to 91 years in age. We successfully retrieved partial sequences of the standard marker gene (nucSSU) in all cases, as well as additional markers (mtSSU, EF1a, and COI) for certain collections. Altogether, 28 genes were recognized in the studied material. In a 33-year-old specimen of Lindbladia tubulina, the assembly reached an N50 of 4.19 kb, enabling the recovery of extended functional loci. The input genomic DNA quantity emerges as the primary determinant of sequencing success. Samples with high DNA yields provide representative amounts of contigs coming confirmedly (matching sequences in the NCBI nucleotide database) or potentially (no-hit fraction) from myxomycetes, regardless of specimen age. In addition to target DNA, we revealed distinct signals of both anthropogenic contamination (human DNA and skin microflora) and natural substrate inhabitants, including oribatid mites and bacteria from dead wood, soil, and grass litter. Thus, even in old collections, metagenomic data still carry information regarding the substrate upon which the myxomycete developed. The results demonstrate that short-read genome skimming may help to integrate historical type material of myxomycetes into contemporary phylogenetic research. This method overcomes the length-dependent limitations of traditional Sanger sequencing, thus providing a roadmap for the future of museomics in myxomycetology.

Amoebozoa

[Number of complaints in cashiers at workplaces with conventional cash registers and those with barcode reader equipment].

115 cash register operators of supermarkets have been studied. Postural complaints are high, compared to office workers. The results obtained by questionnaire and by a rheumatological examination revealed that most cash register operators complain about pains in the back, in the left shoulder and in the left arm. The cashiers working on laser-scanner-systems have the same complaints, but they have significantly less pains in the right arm and the right hand because the cash register is only used infrequently. It seems to be better if cashiers have to do different tasks in a supermarket because the study shows that cashiers who work at different workplaces have significantly less pains then those who work only at the cash register.

Back Pain

Comprehensive plastome variation and RNA editing in Mentha: insights into phylogenetic relationships and candidate DNA barcodes.

INTRODUCTION: Mentha is an economically and medicinally important genus in Lamiaceae, but its taxonomy and species delimitation remain challenging because of frequent hybridization, polyploidy, and marked morphological plasticity. METHODS: In this study, we comparatively analyzed 12 plastomes representing major Mentha species, hybrid taxa, and unresolved accessions, including four newly assembled genomes, to characterize plastome structure, repeat composition, sequence divergence, phylogenetic relationships, and plastid RNA editing. The M. arvensis plastome and RNA-seq datasets originated from independent Swiss and Indian accessions, respectively. RESULTS: The plastomes were highly conserved in overall organization, ranging from 151,824 to 152,154 bp and displaying the typical quadripartite structure. Gene content and order were largely stable across taxa, with only minor variation likely associated with annotation differences at IR/SC boundary regions. Codon usage analysis revealed a clear bias toward A/U-ending synonymous codons, and most shared protein-coding genes showed low Ka/Ks ratios, indicating predominant purifying selection. Repeat analyses showed that simple sequence repeats were mainly composed of A/T-rich mononucleotide motifs, whereas long repeats were concentrated in the 30-40 bp size class. Comparative analyses identified six hypervariable regions, namely ccsA-ndhD, ycf1, ndhD, rpl32-trnL-UAG, rbcL-accD, and petA-psbJ, which represent promising candidate plastid markers for species discrimination. Phylogenetic analysis based on complete plastomes provided strong support for relationships among the sampled taxa and recovered a close affinity among M. aquatica, M. arvensis, and M. canadensis. In addition, RNA-seq analysis of M. arvensis identified 17 candidate plastid RNA editing sites, most of which were C-to-U conversions and nonsynonymous events. DISCUSSION: Together, these results expand plastid genomic resources for Mentha and provide a useful framework for phylogenetic inference, species identification, and future germplasm utilization.

RNA editing

Shedding dynamics of a DNA virus population during acute and long-term persistent infection.

Although much is known of the molecular mechanisms of virus infection within cells, substantially less is understood about within-host infection. Such knowledge is key to understanding how viruses take up residence and transmit infectious virus, in some cases throughout the life of the host. Here, using murine polyomavirus (muPyV) as a tractable model, we monitor parallel infections of thousands of differentially barcoded viruses within a single host. In individual mice, we show that numerous viruses (>2600) establish infection and are maintained for long periods post-infection. Strikingly, a low level of many different barcodes is shed in urine at all times post-infection, with a minimum of at least 80 different barcodes present in every sample throughout months of infection. During the early acute phase, bulk shed virus genomes derive from numerous different barcodes. This is followed by long term persistent infection detectable in diverse organs. Consistent with limited productive exchange of virus genomes between organs, each displays a unique pattern of relative barcode abundance. During the persistent phase, constant low-level shedding of typically hundreds of barcodes is maintained but is overlapped with rare, punctuated shedding of high amounts of one or a few individual barcodes. In contrast to the early acute phase, these few infrequent highly shed barcodes comprise the majority of bulk shed genomes observed during late times of persistent infection, contributing to a stark decrease in bulk barcode diversity that is shed over time. These temporally shifting patterns, which are conserved across hosts, suggest that polyomaviruses balance continuous transmission potential with reservoir-driven high-level reactivation. This offers a mechanistic basis for polyomavirus ubiquity and long-term persistence, which are typical of many DNA viruses.

Animals

Plant species identification by genome skimming across the vascular plant tree of life.

Accurate species identification is essential for biodiversity conservation and sustainable use, yet standard plant DNA barcoding often fails to achieve species-level resolution. We present a large-scale empirical evaluation of genome skimming as a tool to improve plant species discrimination. Using standardised data from 1969 individuals representing 475 species from 32 genera across major lineages of the vascular plant tree of life, we compare conventional plastid + internal transcribed spacer (ITS) barcodes with genome skimming approaches. Standard barcoding using rbcL, matK, trnH-psbA and ITS resolved about half of species (49.3%), with six genera showing <&#x2009;25% species discrimination. By contrast, genome skimming enabled the recovery of complete plastid genomes, yielding 57.6% species discrimination. It also generated sufficient nuclear genomic data for additional resolution from k-mer analysis, achieving 66.8% species discrimination - an average gain of 17.5% over standard barcodes - while eliminating cases of extreme failure (<&#x2009;25% resolution). The recovery of complete plastomes and ribosomal DNAs from genome skims also ensures backward compatibility with existing barcode datasets. Our results demonstrate that genome skimming provides data that substantially improves species-level resolution across diverse plant lineages and offers a scalable, high-throughput approach for building comprehensive reference resources to support global biodiversity initiatives.

DNA Barcoding, Taxonomic

Homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes in somatic cells associate with each other, a phenomenon known as somatic homolog pairing. Unlike in meiosis, where homology is read out at the level of DNA sequence complementarity, somatic homolog pairing takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, presumably mediated by different proteins that bind to these different regions. Here we consider an alternative model, which we term the "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. An important component of this model is that the buttons are non-uniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a non-homolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. We investigated several types of barcodes and examined their effect on pairing fidelity. We found that high fidelity homolog recognition can be achieved by arranging chromosome pairing buttons according to an actual industrial barcode used for warehouse sorting. By simulating randomly generated non-uniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Preprint

Molecular identification of Hymenopteran insects collected by using Malaise traps from Hazarganji Chiltan National Park Quetta, Pakistan.

The order Hymenoptera holds great significance for humans, particularly in tropical and subtropical regions, due to its role as a pollinator of wild and cultivated flowering plants, parasites of destructive insects and honey producers. Despite this importance, limited attention has been given to the genetic diversity and molecular identification of Hymenopteran insects in most protected areas. This study provides insights into the first DNA barcode of Hymenopteran insects collected from Hazarganji Chiltan National Park (HCNP) and contributes to the global reference library of DNA barcodes. A total of 784 insect specimens were collected using Malaise traps, out of which 538 (68.62%) specimens were morphologically identified as Hymenopteran insects. The highest abundance of species of Hymenoptera (133/538, 24.72%) was observed during August and least in November (16/538, 2.97%). Genomic DNA extraction was performed individually from 90/538 (16.73%) morphologically identified specimens using the standard phenol-chloroform method, which were subjected separately to the PCR for their molecular confirmation via the amplification of cytochrome c oxidase subunit 1 (cox1) gene. The BLAST analyses of obtained sequences showed 91.64% to 100% identities with related sequences and clustered phylogenetically with their corresponding sequences that were reported from Australia, Bulgaria, Canada, Finland, Germany, India, Israel, and Pakistan. Additionally, total of 13 barcode index numbers (BINs) were assigned by Barcode of Life Data Systems (BOLD), out of which 12 were un-unique and one was unique (BOLD: AEU1239) which was assigned for Anthidium punctatum. This indicates the potential geographical variation of Hymenopteran population in HCNP. Further comprehensive studies are needed to molecularly confirm the existing insect species in HCNP and evaluate their impacts on the environment, both as beneficial (for example, pollination, honey producers and natural enemies) and detrimental (for example, venomous stings, crop damage, and pathogens transmission).

Humans

SpaceBar enables clone tracing in spatial transcriptomic data.

We report a cellular barcoding strategy, SpaceBar, that enables simultaneous clone tracing and spatial transcriptomics profiling. Our approach uses a library of 96 synthetic barcode sequences that can be robustly detected by imaging based spatial transcriptomics (seqFISH), delivered such that each cell is labeled with a combination of barcodes. We used these barcodes to label melanoma cells in a tumor xenograft model and profiled both clone identity and spatial gene expression in situ. We developed a gene scoring metric that quantifies how strongly gene expression is driven by intrinsic cellular cues or extrinsic environmental signals. Our framework distinguishes between clonal dynamics and environmentally-driven transcriptional regulation in complex tissue contexts.

Journal Article

Deciphering Cell Fate and Clonal Dynamics via Integrative Single-Cell Lineage Modeling.

Through natural or synthetic lineage barcodes, single-cell technologies now enable the joint measurement of molecular states and clonal identities, providing an unprecedented opportunity to study cell fate and dynamics. Yet, most computational methods for inferring cell development and differentiation rely exclusively on transcriptional similarity, overlooking the lineage information encoded by lineage barcodes. This limitation is exemplified by T cells, where subtle transcriptional differences mark divergent fates with distinct biological activity. Single-cell RNA and matched TCR sequencing is now ubiquitous in the analysis of clinical samples, where the TCR sequence provides an endogenous clonal barcode and could reveal clonal T cell responses. We present Clonotrace, a computational framework that jointly models gene expression and clonotype information to infer cell state transitions and fate biases with higher fidelity. While motivated by challenges in analyzing T cell populations, especially in the tumor microenvironment and immunotherapy settings, Clonotrace is broadly applicable to any lineage-barcoded single-cell dataset. Across diverse systems including T cells, hematopoietic differentiation, and cancer therapy resistance models, Clonotrace reveals differentiation hierarchies, distinguishes unipotent from multipotent states, and identifies candidate fate-determining genes driving lineage commitment.

Journal Article

Pooled PPIseq: Screening the SARS-CoV-2 and human interface with a scalable multiplexed protein-protein interaction assay platform.

Protein-Protein Interactions (PPIs) are a key interface between virus and host, and these interactions are important to both viral reprogramming of the host and to host restriction of viral infection. In particular, viral-host PPI networks can be used to further our understanding of the molecular mechanisms of tissue specificity, host range, and virulence. At higher scales, viral-host PPI screening could also be used to screen for small-molecule antivirals that interfere with essential viral-host interactions, or to explore how the PPI networks between interacting viral and host genomes co-evolve. Current high-throughput PPI assays have screened entire viral-host PPI networks. However, these studies are time consuming, often require specialized equipment, and are difficult to further scale. Here, we develop methods that make larger-scale viral-host PPI screening more accessible. This approach combines the mDHFR split-tag reporter with the iSeq2 interaction-barcoding system to permit massively-multiplexed PPI quantification by simple pooled engineering of barcoded constructs, integration of these constructs into budding yeast, and fitness measurements by pooled cell competitions and barcode-sequencing. We applied this method to screen for PPIs between SARS-CoV-2 proteins and human proteins, screening in triplicate >180,000 ORF-ORF combinations represented by >1,000,000 barcoded lineages. Our results complement previous screens by identifying 74 putative PPIs, including interactions between ORF7A with the taste receptors TAS2R41 and TAS2R7, and between NSP4 with the transmembrane KDELR2 and KDELR3. We show that this PPI screening method is highly scalable, enabling larger studies aimed at generating a broad understanding of how viral effector proteins converge on cellular targets to effect replication.

Humans

Deciphering Cell Fate and Clonal Dynamics via Integrative Single-Cell Lineage Modeling.

Through natural or synthetic lineage barcodes, single-cell technologies now enable the joint measurement of molecular states and clonal identities, providing an unprecedented opportunity to study cell fate and dynamics. Yet, most computational methods for inferring cell development and differentiation rely exclusively on transcriptional similarity, overlooking the lineage information encoded by lineage barcodes. This limitation is exemplified by T cells, where subtle transcriptional differences mark divergent fates with distinct biological activity. Single-cell RNA and matched TCR sequencing is now ubiquitous in the analysis of clinical samples, where the TCR sequence provides an endogenous clonal barcode and could reveal clonal T cell responses. We present Clonotrace, a computational framework that jointly models gene expression and clonotype information to infer cell state transitions and fate biases with higher fidelity. While motivated by challenges in analyzing T cell populations, especially in the tumor microenvironment and immunotherapy settings, Clonotrace is broadly applicable to any lineage-barcoded single-cell dataset. Across diverse systems including T cells, hematopoietic differentiation, and cancer therapy resistance models, Clonotrace reveals differentiation hierarchies, distinguishes unipotent from multipotent states, and identifies candidate fate-determining genes driving lineage commitment.

Journal Article

A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.

barcoding

A genetic atlas for the butterflies of continental Canada and United States.

Multi-locus genetic data for phylogeographic studies is generally limited in geographic and taxonomic scope as most studies only examine a few related species. The strong adoption of DNA barcoding has generated large datasets of mtDNA COI sequences. This work examines the butterfly fauna of Canada and United States based on 13,236 COI barcode records derived from 619 species. It compiles i) geographic maps depicting the spatial distribution of haplotypes, ii) haplotype networks (minimum spanning trees), and iii) standard indices of genetic diversity such as nucleotide diversity (&#x3c0;), haplotype richness (H), and a measure of spatial genetic structure (GST). High intraspecific genetic diversity and marked spatial structure were observed in the northwestern and southern North America, as well as in proximity to mountain chains. While species generally displayed concordance between genetic diversity and spatial structure, some revealed incongruence between these two metrics. Interestingly, most species falling in this category shared their barcode sequences with one at least other species. Aside from revealing large-scale phylogeographic patterns and shedding light on the processes underlying these patterns, this work also exposed cases of potential synonymy and hybridization.

Animals

Modeling homologous chromosome recognition via nonspecific interactions.

In many organisms, most notably Drosophila, homologous chromosomes associate in somatic cells, a phenomenon known as somatic pairing, which takes place without double strand breaks or strand invasion, thus requiring some other mechanism for homologs to recognize each other. Several studies have suggested a "specific button" model, in which a series of distinct regions in the genome, known as buttons, can associate with each other, mediated by different proteins that bind to these different regions. Here, we use computational modeling to evaluate an alternative "button barcode" model, in which there is only one type of recognition site or adhesion button, present in many copies in the genome, each of which can associate with any of the others with equal affinity. In this model, buttons are nonuniformly distributed, such that alignment of a chromosome with its correct homolog, compared with a nonhomolog, is energetically favored; since to achieve nonhomologous alignment, chromosomes would be required to mechanically deform in order to bring their buttons into mutual register. By simulating randomly generated nonuniform button distributions, many highly effective button barcodes can be easily found, some of which achieve virtually perfect pairing fidelity. This model is consistent with existing literature on the effect of translocations of different sizes on homolog pairing. We conclude that a button barcode model can attain highly specific homolog recognition, comparable to that seen in actual cells undergoing somatic homolog pairing, without the need for specific interactions. This model may have implications for how meiotic pairing is achieved.

Animals

Integration of Imaging-based and Sequencing-based Spatial Omics Mapping on the Same Tissue Section via DBiTplus.

Spatially mapping the transcriptome and proteome in the same tissue section can significantly advance our understanding of heterogeneous cellular processes and connect cell type to function. Here, we present Deterministic Barcoding in Tissue sequencing plus (DBiTplus), an integrative multi-modality spatial omics approach that combines sequencing-based spatial transcriptomics and image-based spatial protein profiling on the same tissue section to enable both single-cell resolution cell typing and genome-scale interrogation of biological pathways. DBiTplus begins with in situ reverse transcription for cDNA synthesis, microfluidic delivery of DNA oligos for spatial barcoding, retrieval of barcoded cDNA using RNaseH, an enzyme that selectively degrades RNA in an RNA-DNA hybrid, preserving the intact tissue section for high-plex protein imaging with CODEX. We developed computational pipelines to register data from two distinct modalities. Performing both DBiT-seq and CODEX on the same tissue slide enables accurate cell typing in each spatial transcriptome spot and subsequently image-guided decomposition to generate single-cell resolved spatial transcriptome atlases. DBiTplus was applied to mouse embryos with limited protein markers but still demonstrated excellent integration for single-cell transcriptome decomposition, to normal human lymph nodes with high-plex protein profiling to yield a single-cell spatial transcriptome map, and to human lymphoma FFPE tissue to explore the mechanisms of lymphomagenesis and progression. DBiTplusCODEX is a unified workflow including integrative experimental procedure and computational innovation for spatially resolved single-cell atlasing and exploration of biological pathways cell-by-cell at genome-scale.

Journal Article