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PubMed2025

ChemPerturb-seq screen identifies a small molecule cocktail enhancing human beta cell survival after subcutaneous transplantation.

Traditional chemical screens have focused on a single assay per screen, making them labor intensive and costly. Here, we combined a chemical screen with single-cell RNA sequencing (scRNA-seq) to perform Chemical Perturb-seq (ChemPerturb-seq), enabling a systematic analysis of the molecular changes of human beta cells upon individual small molecule treatments. Using this platform, we performed an in vivo barcoded screen and discovered a small molecule cocktail, including beta-lipotropin 61-91, insulin growth factor-1, and prostaglandin E2, with which preconditioning human beta cells and primary islets significantly enhanced function and survival when transplanted subcutaneously to female, but not to male, mice. We identified two additional molecules, serotonin and histamine, that promote islet function when transplanted subcutaneously to male mice using ChemPerturb-seq. Such small molecule cocktails could be applied to improve the current FDA-approved islet transplantation procedure. Finally, we developed an artificial intelligence (AI)-powered website, ChemPerturbDB, which provides user-friendly open access analysis of the extensive ChemPerturb-seq dataset.

Humans↗
PubMed2026

Comparative chloroplast genomics of six Bupleurum (Apiaceae) accessions: candidate barcodes, phylogeny based on available plastomes, and candidate RNA-editing sites.

INTRODUCTION: Bupleurum L. (Apiaceae), a taxonomically intricate genus of about 190 species and a source of Radix Bupleuri (Chai Hu), is difficult to discriminate because of convergent morphology, infraspecific variation, and limited genomic sampling. This study aimed to characterize plastome variation, identify and validate candidate molecular markers, reconstruct plastid phylogenetic relationships, and assess candidate plastid RNA-editing sites in Bupleurum. METHODS: We assembled six plastomes from subgenus Bupleurum, screened 51 Bupleurum plastomes for diagnostic loci, reconstructed whole-plastome and partitioned protein-coding-sequence phylogenies, and predicted plastid C-to-U RNA-editing candidates across the six newly assembled plastomes using a PREP-Cp-compatible workflow. Candidate barcode performance was evaluated against the reference plastome phylogenies, and codon-based models were used to test for positive selection. RESULTS: The plastomes were 154,496-155,778 bp with the canonical quadripartite structure and GC contents of 37.67-37.73%. Gene content was stable (131-132 genes; 86-87 protein-coding genes); B. falcatum subsp. cernuum lacked ycf15 but contained an additional inverted-repeat-associated ycf1 annotation. A/U-ending synonymous codons were favoured. Finite pairwise Ka/Ks estimates were below 1 for most genes, and site-specific codon models detected no positive selection. Each plastome contained 55-61 pure microsatellites, dominated by A/T mononucleotide motifs. MarkerSeek ranked 265 features and identified atpF-atpH, petA-psbJ, rpl32-trnL-UAG, and ycf1 as leading candidate barcodes. ycf1 recovered 38 of 41 nodes strongly supported by both reference trees, whereas a partitioned four-locus analysis recovered 40 of 41 and distinguished all 51 accession sequences. However, only one of seven multi-accession operational binomial groups was monophyletic, and only one showed a positive local barcode gap. The whole-plastome phylogeny recovered Bupleurum as monophyletic relative to Chamaesium. The two sampled Penninervia accessions occupied early-diverging positions without forming an exclusive clade. B. falcatum subsp. cernuum was sister to B. ranunculoides, with B. ranunculoides subsp. telonense sister to that pair. A partitioned 74-CDS analysis recovered the same key relationships and 45 of 50 internal bipartitions. Across the six newly assembled plastomes, 57-63 nonsynonymous C-to-U candidates were predicted per accession (367 total) in 21-22 genes; 269 affected the second codon position and 98 the first. DISCUSSION: Bupleurum plastomes are structurally conservative but retain localised divergence useful for marker development. Concordant whole-plastome and CDS genealogies support genus monophyly, whereas sparse Penninervia sampling and maternal plastid inheritance preclude rejecting traditional subgeneric classification. The predicted RNA-editing sites represent candidates for future experimental validation rather than an established Bupleurum editome. These genomic resources support authentication, conservation, and evolutionary research in Bupleurum.

Apiaceae↗
PubMed2026

RUMINA: high-throughput deduplication of unique molecular identifiers for amplicon and whole-genome sequencing with enhanced error correction.

MOTIVATION: Unique molecular identifiers (UMIs) are widely used in next-generation sequencing to enable accurate molecular counting and error correction. However, challenges remain in accurately collapsing UMI clusters, especially when read counts are low or sparse read clusters arise from barcode sequencing errors. RESULTS: We present RUMINA, a Rust-based pipeline for UMI-aware deduplication and error correction, optimized for both amplicon and shotgun sequencing. RUMINA supports multiple UMI cluster strategies, alongside majority-rule read selection independent of mapping quality, as well as discrete handling of 1-2 read clusters, paired-end merging, and read-length stratification. Benchmarking using simulated HIV population sequencing data and real-world iCLIP and TCR datasets showed that RUMINA improves ultra-low frequency SNV detection (0.01%-1%), reduces false positives, enhances reproducibility, and processes sequencing data up to 10-fold faster than existing tools. By integrating UMI- and sequence-level correction in a high-performance framework, RUMINA offers a fast, scalable, and robust solution for UMI-enabled sequencing workflows. AVAILABILITY AND IMPLEMENTATION: RUMINA is implemented in Rust and distributed as open-source code and precompiled binaries. Source code and installation instructions are available at https://github.com/greninger-lab/rumina. Documentation associated with this manuscript is available at https://github.com/greninger-lab/rumina_paper.

High-Throughput Nucleotide Sequencing↗
PubMed2000

Cryptography with DNA binary strands.

Biotechnological methods can be used for cryptography. Here two different cryptographic approaches based on DNA binary strands are shown. The first approach shows how DNA binary strands can be used for steganography, a technique of encryption by information hiding, to provide rapid encryption and decryption. It is shown that DNA steganography based on DNA binary strands is secure under the assumption that an interceptor has the same technological capabilities as sender and receiver of encrypted messages. The second approach shown here is based on steganography and a method of graphical subtraction of binary gel-images. It can be used to constitute a molecular checksum and can be combined with the first approach to support encryption. DNA cryptography might become of practical relevance in the context of labelling organic and inorganic materials with DNA 'barcodes'.

Computer Communication Networks↗
PubMed2024

Directed evolution of engineered virus-like particles with improved production and transduction efficiencies.

Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of proteins and RNAs, including gene editing agents. We report a system for the laboratory evolution of eVLPs that enables the discovery of eVLP variants with improved properties. The system uses barcoded guide RNAs loaded within DNA-free eVLP-packaged cargos to uniquely label each eVLP variant in a library, enabling the identification of desired variants following selections for desired properties. We applied this system to mutate and select eVLP capsids with improved eVLP production properties or transduction efficiencies in human cells. By combining beneficial capsid mutations, we developed fifth-generation (v5) eVLPs, which exhibit a 2-4-fold increase in cultured mammalian cell delivery potency compared to previous-best v4 eVLPs. Analyses of v5 eVLPs suggest that these capsid mutations optimize packaging and delivery of desired ribonucleoprotein cargos rather than native viral genomes and substantially alter eVLP capsid structure. These findings suggest the potential of barcoded eVLP evolution to support the development of improved eVLPs.

Humans↗
PubMed2004

Evaluation of molecular typing methods for Escherichia coli O157:H7 isolates from cattle, food, and humans.

Escherichia coli O157:H7, a Shiga toxin-producing E. coli, has been the causative agent of many cases of severe, often life-threatening foodborne illness. Because of the importance of E. coli O157:H7 to public health, many molecular typing methods have been developed to determine its transmission routes and source of infection during epidemiological investigations. Pulsed-field gel electrophoresis (PFGE) is currently used by public health organizations to track infections of E. coli O157:H7 and other foodborne pathogens. In this study, we compared the ability of PFGE, multilocus sequence typing (MLST), and repetitive-element PCR (Rep-PCR) to distinguish among 92 E. coli O157:H7 isolates from cattle, food, and infected humans. Several virulence genes, including the intimin gene (eaeA), the hemolysin gene (hlyA), and the H7 fimbrial gene (fliC), and a housekeeping gene for beta-glucuronidase (uidA) were included in MLST. Rep-PCR reactions were performed using a commercially available typing kit (Bacterial Barcodes Inc., Houston, Tex.) with the provided Uprime-RI primer set. Results of the study indicated that PFGE provided the most discrimination among the techniques, identifying 72 distinct PFGE profiles for the isolates; Rep-PCR elucidated 14 different profiles, whereas MLST generated five profiles. Additionally, there did not appear to be any correlation among the typing methods examined in this study. Therefore, to date, PFGE remains the technique of choice for molecular subtyping of E. coli O157:H7.

Animals↗
PubMed2003

Nanodiagnostics: application of nanotechnology in molecular diagnostics.

Nanotechnology extends the limits of molecular diagnostics to the nanoscale. Nanotechnology-on-a-chip is one more dimension of microfluidic/lab-on-a-chip technology. Biological tests measuring the presence or activity of selected substances become quicker, more sensitive and more flexible when certain nanoscale particles are put to work as tags or labels. Magnetic nanoparticles, bound to a suitable antibody, are used to label specific molecules, structures or microorganisms. Magnetic immunoassay techniques have been developed in which the magnetic field generated by the magnetically labeled targets is detected directly with a sensitive magnetometer. Gold nanoparticles tagged with short segments of DNA can be used for detection of genetic sequence in a sample. Multicolor optical coding for biological assays has been achieved by embedding different-sized quantum dots into polymeric microbeads. Nanopore technology for analysis of nucleic acids converts strings of nucleotides directly into electronic signatures. DNA nanomachines can function as biomolecular detectors for homogeneous assays. Nanobarcodes, submicrometer metallic barcodes with striping patterns prepared by sequential electrochemical depositon of metal, show differential reflectivity of adjacent stripes enabling identification of the striping patterns by conventional light microscopy. All this has applications in population diagnostics and in point-of-care hand-held devices.

Animals↗
PubMed2026

Comparative Analysis of Chloroplast Genomes Reveals Molecular Evolution and Phylogenetic Relationships in Fraxinus (Fraxinus mandshurica).

Fraxinus mandshurica (Manchurian ash) is an ecologically and economically valuable hardwood tree native to Northeast Asia, yet its genomic resources remain limited. We assembled its complete chloroplast (cp) genome (155,559 bp) using hybrid PacBio and Illumina sequencing and performed comparative, phylogenetic, and evolutionary analyses. The cp genome exhibits a typical quadripartite structure encoding 132 gene copies, comprising 114 unique genes (80 protein-coding, 30 tRNA, and 4 rRNA genes), with 18 genes duplicated in the inverted repeat (IR) regions. Simple sequence repeat analysis revealed dominance of mononucleotide A/T repeats. Phylogenetic analysis of 53 complete cp genomes strongly supported the monophyly of Oleaceae and resolved F. mandshurica as sister to the North American F. nigra, consistent with previously proposed Miocene intercontinental dispersal scenarios between East Asia and North America. Most protein-coding genes were under strong purifying selection (Ka/Ks << 1), whereas petB, rpl2, and several ndh genes showed elevated Ka/Ks values that are suggestive of altered selective constraint but are based on very few substitutions and are therefore not, on their own, evidence of positive selection. Nucleotide diversity (Pi) analysis identified 15 hypervariable intergenic spacers (mean Pi = 0.067), among which trnM-CAU-rps14, ndhJ-ndhK, and petL-petG represent promising candidate barcode regions requiring further validation. This study provides a high-quality, fully annotated cp genome of F. mandshurica and a valuable genomic resource for future phylogenetic, population genetic, and conservation studies of this important genus.

Fraxinus↗
PubMed2004

Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator.

Astraptes fulgerator, first described in 1775, is a common and widely distributed neotropical skipper butterfly (Lepidoptera: Hesperiidae). We combine 25 years of natural history observations in northwestern Costa Rica with morphological study and DNA barcoding of museum specimens to show that A. fulgerator is a complex of at least 10 species in this region. Largely sympatric, these taxa have mostly different caterpillar food plants, mostly distinctive caterpillars, and somewhat different ecosystem preferences but only subtly differing adults with no genitalic divergence. Our results add to the evidence that cryptic species are prevalent in tropical regions, a critical issue in efforts to document global species richness. They also illustrate the value of DNA barcoding, especially when coupled with traditional taxonomic tools, in disclosing hidden diversity.

Animal Feed↗
PubMed2025

Genome-wide phylogeny reshapes our understanding of the evolution of deep-sea dragonfishes, bristlemouths, viperfishes, and allies (Stomiiformes).

BACKGROUND: The evolutionary relationships within Stomiiformes, a diverse order of deep-sea fishes dominating the mesopelagic and bathypelagic zones, remain contentious due to conflicting morphological and molecular evidence. These fishes, comprising 464 species across four traditionally recognized families (Gonostomatidae, Sternoptychidae, Phosichthyidae, and Stomiidae), exhibit remarkable adaptations such as bioluminescence, ultra-black pigmentation, and extreme jaw morphologies. Their global abundance and ecological significance, including contributions to the biological carbon pump, underscores the need to resolve their phylogeny amid escalating threats from climate change and human activities. RESULTS: We conducted the most comprehensive phylogenomic analysis of Stomiiformes to date, integrating 936 nuclear loci from 60 species and an expanded dataset of 135 species with mitochondrial sequences from publicly available repositories such as the Barcode of Life Data Systems (BOLD) database. We used maximum likelihood and coalescent-based approaches to assess family monophyly and relationships, including extensive quality control to address contamination in public databases. Our analyses reveal unstable tree topologies and complex evolutionary histories that challenge traditional classifications, while our quality control analyses identified 29% of BOLD sequences as misidentified or contaminated, emphasizing rigorous curation for deep-sea taxa. Congruent with a recent taxonomic treatment of Stomiiformes, the families Phosichthyidae and Gonostomatidae exhibit polyphyly and paraphyly, respectively, while subfamilies within Stomiidae are extensively non-monophyletic, leading us to recommend their abandonment. We propose the recognition of eight monophyletic families: Vinciguerriidae, Diplophidae, Gonostomatidae, Yarrellidae, Ichthyococcidae, Phosichthyidae, Sternoptychidae, and Stomiidae, supported by robust molecular and morphological evidence. CONCLUSIONS: This revised classification reflects the morphological and ecological diversity of Stomiiformes, aligning with their evolutionary diversification in the deep sea. Our phylogenomic framework resolves longstanding systematic uncertainties and highlights the power of genome-wide data in tackling taxonomically challenging clades. These findings provide a foundation for understanding deep-sea fish diversification and assessing the potential ecological drivers for their evolutionary diversity.

Animals↗
PubMed2025

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↗
PubMed1993

Digital codes from hypervariable tandemly repeated DNA sequences in the Plasmodium falciparum circumsporozoite gene can genetically barcode isolates.

DNA typing systems currently used in parasitology involve either hybridising Southern blots with repetitive sequence probes or amplifying genomic sequences using the polymerase chain reaction (PCR). Both such approaches assay allelic length variation, usually in unexpressed tandemly repeated DNA sequences. Where an appropriate target locus exists, an alternative PCR-based strategy which reveals allelic sequence variation in tandemly repeated DNA offers a more accurate and internally controlled assay. We describe such a strategy for the rapid extraction of information on tandem repeat sequence variation from hypervariable alleles, and apply it to the Plasmodium falciparum CS gene. The extreme variability of such DNA 'barcodes' can be used to identify parasite stocks and lineages. This system is also potentially useful for population genetic and epidemiological studies since it offers the possibility of following the spread of distinctively marked parasite genotypes in samples taken from infected individuals.

Animals↗
PubMed2004

Identification of Birds through DNA Barcodes.

Short DNA sequences from a standardized region of the genome provide a DNA barcode for identifying species. Compiling a public library of DNA barcodes linked to named specimens could provide a new master key for identifying species, one whose power will rise with increased taxon coverage and with faster, cheaper sequencing. Recent work suggests that sequence diversity in a 648-bp region of the mitochondrial gene, cytochrome c oxidase I (COI), might serve as a DNA barcode for the identification of animal species. This study tested the effectiveness of a COI barcode in discriminating bird species, one of the largest and best-studied vertebrate groups. We determined COI barcodes for 260 species of North American birds and found that distinguishing species was generally straightforward. All species had a different COI barcode(s), and the differences between closely related species were, on average, 18 times higher than the differences within species. Our results identified four probable new species of North American birds, suggesting that a global survey will lead to the recognition of many additional bird species. The finding of large COI sequence differences between, as compared to small differences within, species confirms the effectiveness of COI barcodes for the identification of bird species. This result plus those from other groups of animals imply that a standard screening threshold of sequence difference (10x average intraspecific difference) could speed the discovery of new animal species. The growing evidence for the effectiveness of DNA barcodes as a basis for species identification supports an international exercise that has recently begun to assemble a comprehensive library of COI sequences linked to named specimens.

Animals↗
PubMed2026

Assessment of Genetic Diversity and Population Structure on Azadirachta indica A. Juss. in an Urban Metropolitan: Ahmedabad, India.

Azadirachta indica (A. indica) A. Juss., commonly known as Neem, is a valuable multipurpose tree with profound medicinal properties and socioeconomic importance, widely recognized since ancient Ayurvedic times. Despite its prominence, knowledge about its genetic diversity within the metropolitan area of Ahmedabad is limited. This study marks the first in-depth exploration of the genetic diversity and population structure of A. indica in Ahmedabad. The authenticity of the species was validated through DNA barcoding, and a Geographical Information System (GIS) was used to collect the samples. A total of 35 A. indica accessions were analyzed using five Inter Simple Sequence Repeat (ISSR) primers. Genetic diversity and population structure were evaluated using Inter Simple Sequence Repeat (ISSR) markers through polymorphism assessment, clustering, ordination, and Bayesian population structure analyses. ISSRs revealed a high level of polymorphism (75.66%), indicating substantial genetic variability among accessions. An analysis of genetic diversity indices revealed low to moderate diversity (Hs&#x2009;=&#x2009;0.14, Ht&#x2009;=&#x2009;0.217, I&#x2009;=&#x2009;0.217). Analysis of Molecular Variance (AMOVA) analysis depicted 81% variation within the population and 19% among the population. Low to moderate genetic differentiation (Gst&#x2009;=&#x2009;0.319) and moderate gene flow (Nm&#x2009;=&#x2009;1.06) indicated that urban development has not hindered gene flow among populations. Mantel's test revealed a weak but significant correlation between genetic and geographic distances, suggesting limited isolation by distance. The estimated &#x394;K using STRUCTURE exhibited two subpopulations, representing two gene pools for A. indica accessions (K&#x2009;=&#x2009;2). Collectively, these patterns indicate that urbanization has not severely disrupted genetic connectivity in A. indica, reflecting its resilience and adaptive potential in a metropolitan environment. These findings provide pivotal knowledge for further understanding the genetic diversity and population structure of A. indica in one of the fastest-growing cities in India, which can be utilized for new breeding programmes, sustainable development and future conservation strategies around the globe.

India↗
PubMed1996

Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization.

Several hematologic malignancies are associated with specific chromosomal translocations. Because of the dispersed distribution, chromosomal breakpoints may be difficult to detect using molecular techniques. We present a new application of a recently developed method, DNA fiber fluorescence in situ hybridization (fiber FISH), which allows direct visualization and mapping of chromosomal breakpoints. We tested this method for detection of the t(11;14)(q13;q32) translocation in mantle cell lymphoma. In DNA fiber FISH, a series of fluorochrome-labeled DNA probes covering several hundreds of kilobasepairs is hybridized to linear DNA molecules (or fibers) prepared from frozen tissue or intact cells. By using alternate fluorescent colors, a potential breakpoint region is stained in a color barcode pattern. Breaks in this region will split the barcode in two complementary parts, from which the breakpoint position can be derived. We used a 250-kb barcode covering the BCL-1 locus to detect 11q13 breakpoints in 20 well-characterized mantle cell lymphomas. A t(11;14) was shown by cohybridization of these probes with probes for the Ig heavy chain locus at 14q32. In 18 of 20 mantle cell lymphomas, a breakpoint within the 11q13/BCL-1 barcode was shown by the presence of multiple, complementary translocation products. Fusion of 11q13 and 14q32 sequences on single fibers indicating t(11;14)(q13;q32) was found in all 18 breakpoint-positive mantle cell lymphomas. In one additional case, fusion of an intact 11q13 barcode with 14q32 sequences indicated a breakpoint 100 kb centromeric of the major translocation cluster of BCL-1. Within the 120-kb region of BCL-1, breakpoints were widely scattered. This explains why, so far, a BCL-1 breakpoint had been detected by Southern blot analysis in only 10 of 19 cases. DNA fiber FISH analysis showed a t(11;14) in 95% of mantle cell lymphoma. The results indicate that DNA fiber FISH is a rapid, simple, and equally powerful method for detection of clustered and dispersed translocation breakpoints.

Chromosome Aberrations↗
PubMed2025

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↗