Biology subjects

Mansfield, M. J.

Publications and source records attributed to Mansfield, M. J..

11 recordsLinked to original sources

bioRxiv2025

Unlocking the full potential of Oxford Nanopore reads with NOVOLoci

Long-read sequencing technologies and accompanying algorithms have substantially advanced genome assembly. However, there are ongoing challenges in assembling complex genomic regions, particularly those associated with genetic disorders. Moreover, assemblies of non-model organisms are often limited by the amount of DNA that can be extracted, so reducing the data modalities that can be obtained. Here, we introduce NOVOLoci, a haplotype-aware assembler capable of high-quality targeted and whole-genome assemblies, despite the relatively high error rates of Oxford Nanopore Technologies (ONT) data. By adopting a novel seed-extension approach with iterative conflict resolution, it achieves accurate haplotype phasing, thus overcoming a critical limitation of current graph-based assemblers. Benchmarking shows that NOVOLoci consistently outperforms the four leading assembly tools across five clinically relevant genomic disorder loci by delivering accurately phased assemblies with superior contiguity and completeness, even compared with hybrid assemblers that combine PacBio and ONT sequencing reads (nearly triple the N90 value compared with Verkko hybrid). We demonstrate its broader utility in assembling the genome of a highly heterozygous non-model animal Oikopleura dioica, whose small size limits the amount of DNA available from an individual. Using data only from a single ONT flow cell, NOVOLoci assembles a phased diploid genome with high contiguity and a 32-fold increase in N50 values compared with existing methods. NOVOLocis distinct algorithmic strategy demonstrates that substantial advances in genome assembly can still be made. This development brings routine clinical diagnosis of genomic disorders and diploid assembly of non-model organisms with ONT reads a step closer to reality.

bioinformatics↗

bioRxiv2024

Less, but more: new insights from appendicularians on chordate Fgf evolution and the divergence of tunicate lifestyles.

The impact of gene loss on the divergence of taxa and the generation of evolutionary innovations is a fundamental aspect of Evolutionary Biology that remains unclear. Here, using the evolution of the Fibroblast Growth Factors (FGFs) in appendicularians as a case study, we investigate how gene losses have influenced the evolution of chordates, especially the divergence among tunicates. Our work reveals an unprecedented case of massive losses of all Fgf gene subfamilies, except for the Fgf9/16/20 and Fgf11/12/13/14, which in turn suffered two bursts of gene duplications. Phylogenetic inferences and genomic analyses of gene synteny conservation, gene architecture, alternative splicing and protein 3D-structure have allowed us to reconstruct the history of appendicularian Fgf genes in the context of chordate evolution, providing compelling evidence supporting the paracrine secreting functions and the intracellular functions of the Fgf9/16/20 and Fgf11/12/13/14 subfamilies, respectively. Exhaustive analysis of developmental Fgf expression in Oikopleura dioica as a model for appendicularians reveals a paradigmatic case of what could be referred as "less, but more", providing a conceptual evolutionary framework characterized by four associated evolutionary patterns: conservation of ancestral Fgf expression domains; function shuffling between paralogs upon gene loss; innovation of new expression domains after the bursts of Fgf duplications; and the extinction of Fgf functions linked to gene losses. The findings of this work allow us to formulate novel hypotheses about the potential impact of losses and duplications of Fgf genes on the transition from an ancestral ascidian-like biphasic lifestyle to a fully free-living style of appendicularians. These hypotheses include the massive co-option of Fgf genes for the patterning of the oikoblast responsible of the house architecture, and for the development of the tail fin; the recruitment of Fgf11/12/13/14 genes into the evolution of a new mouth, and their role modulating neuronal excitability; the evolutionary innovation of an "anterior tail" FGF signaling mesodermal source upon the loss of retinoic acid signaling; and the potential link between the loss of Fgf7/10/22 and Fgf8/17/18 and the loss of drastic metamorphosis, mesenchymal cells and lack of tail absorption in appendicularians, in contrast to ascidians.

evolutionary biology↗

bioRxiv2024

Tracing Homopolymers in Oikopleura dioica's mitogenome

Oikopleura dioica is a planktonic tunicate (Appendicularia class) found extensively across the marine waters of the globe. The genome of a single male individual collected from Okinawa, Japan was sequenced using the single-molecule PacBio Hi-Fi method and assembled with NOVOLoci. The mitogenome is 39,268 bp long, featuring a large control region of around 22,000 bp. We annotated the proteins atp6, cob, cox1, cox2, cox3, nad1, nad4 and nad5, and found one more open reading frame that did not match any known gene. This study marks the first complete mitogenome assembly for an appendicularian, and reveals that A and T homopolymers cumulatively account for nearly half of its length. This reference sequence will be an asset for environmental DNA and phylogenetic studies. http://dx.doi.org/10.1364/ao.XX.XXXXXX

genomics↗

bioRxiv2023

Extreme genome scrambling in cryptic Oikopleura dioica species

Genes are not randomly distributed throughout chromosomes. How gene order evolves and how selective constraints act to preserve or vary gene order, both at the macrosyntenic level of whole chromosomes or microsyntenic level of gene blocks, are central questions of evolutionary biology and genomics that remain largely unsolved. Here, after sequencing several genomes of the appendicularian tunicate Oikopleura dioica from different locations around the globe, we show an unprecedented amount of genome scrambling in animals with no obvious morphological differences, consistent with cryptic speciation. Our assemblies suggest that all members of this clade possess a common 3-chromosome karyotype, and that different species largely preserve gene content, despite the presence of thousands of rearrangements in gene order. The movements of genes are largely restricted to chromosome arms and sex-specific regions, which appear to be the primary unit of macrosynteny conservation, and examples of these within-arm movements can be seen in the Hox and Fgf gene families. Our approach employing whole-genome alignments demonstrates that segments containing protein-coding elements tend to be preserved at the microsyntenic scale, consistent with strong purifying selection, with appreciably less preservation of non-coding elements. Unexpectedly, scrambling did not preserve operon structure across species, suggesting an absence of selective pressure to maintain operon structure. As well, genome scrambling does not occur uniformly across all chromosomes, as short chromosome arms possess shorter genes, smaller operons, more breakpoints, and elevated dN/dS values compared to long chromosome arms. Estimation of divergence times among the cryptic O. dioica lineages yielded an estimated breakpoint accumulation rate of 6 to 25 breakpoints per megabase per million years, which is an order of magnitude higher than the rates for other ascidian tunicates or Drosophila species. Therefore, O. dioica appears to be an attractive animal system to unravel the mechanisms that underlie gene order and synteny conservation, as well as exploring the limits of genome scrambling without an apparent impact on phenotypic evolution.

genomics↗

bioRxiv2022

Identification of divergent botulinum neurotoxin homologs in Paeniclostridium ghonii

Botulinum neurotoxins (BoNTs) are the most potent family of toxins known to science. Bioinformatic studies in recent years have revealed that they are members of a broader toxin family, with an increasing number of divergent homologs identified in genomes of organisms outside of the Clostridium genus. Here, we report the identification of two putative divergent BoNT-like homologs in the genomes of two strains of Paeniclostridium ghonii. We designated them PG-toxin 1 (PGT1) and PG-toxin 2 (PGT2), which share ~54% protein sequence identity. Unlike any other known BoNT homologs, PGT1 and PGT2 are composed of two separate subunits encoded on two neighboring genes: one encoding the protease domain (light chain, LC) with a conserved HExxH motif, and the second encoding the heavy-chain (HC) containing the putative translocation domain and receptor-binding domain. Phylogenetic analysis of both the LC and HC reveal that it is a divergent member of the lineage of BoNT that also includes BoNT/X, BoNT/En and the insecticidal PMP1. The gene clusters harboring PGT1 and PGT2 also include a putative insecticidal delta-endotoxin, Cry8Ea1, as well as putative endolysin and bacteriocin genes that may facilitate lytic toxin secretion, suggesting a possibility that this gene cluster might serve an insecticidal purpose.

bioinformatics↗

bioRxiv2022

The cosmopolitan appendicularian Oikopleura dioica reveals hidden genetic diversity around the globe

Appendicularian tunicates are some of the most abundant mesozooplankton organisms with key roles in marine trophic webs and global carbon flux. Like most appendicularians with cosmopolitan distributions, Oikopleura dioica Fol, 1872 is considered a single species worldwide based on morphological features that distinguish them from other appendicularians. Despite their abundance however, there are still only [~]70 described appendicularian species, compared with over 2,800 ascidian tunicates. Here we perform a molecular phylogenetic, morphological, and reproductive assessment of O. dioica specimens collected from the Ryukyu Archipelago, mainland Japan, and Europe. The specimens are morphologically very similar, with only detailed examination of the oikoplastic epithelium and quantitative measurements revealing minor distinguishing characteristics. Phylogenetic analyses of the ribosomal gene loci and mitochondrial cytochrome oxidase I (COI) gene strongly indicate that they form three separate genetic clades despite their morphological similarities. Finally, in vitro crosses between the Ryukyu and mainland Japanese specimens show total prezygotic reproductive isolation. Our results reveal that the current taxonomic O. dioica classification likely hides multiple cryptic species, highlighting the genetic diversity and complexity of their population structures. Cryptic organisms are often hidden under a single species name because their morphological similarities make them difficult to disinguish and their correct identification is fundamental to understanding Earths biodiversity. O. dioica is an attractive model to understand how morphological conservation can be maintained despite pronounced genetic divergence.

evolutionary biology↗

bioRxiv2022

Ancient Clostridium DNA and variants of tetanus neurotoxins associated with human archaeological remains

The analysis of microbial genomes from human archaeological samples offers a historic snapshot of ancient pathogens and provides insights into the origins of modern infectious diseases. Here, through a large-scale metagenomic analysis of archeological samples, we discovered bacterial species related to modern-day Clostridium tetani, which produces the tetanus neurotoxin (TeNT) and causes the disease tetanus. We assembled draft genomes from 38 distinct human archeological samples spanning five continents and dating to as early as ~4000 BCE. These genomes had varying levels of completeness and a subset of them displayed hallmarks of ancient DNA damage. While 24 fall into known C. tetani clades, phylogenetic analysis revealed novel C. tetani lineages, as well as two novel Clostridium species ("Clostridium sp. X and Y") closely related to C. tetani. Within these genomes, we found 13 TeNT variants with unique substitution profiles, including a subgroup of TeNT variants found exclusively in ancient samples from South America. We experimentally tested a TeNT variant selected from a ~6000-year-old Chilean mummy sample and found that it induced tetanus muscle paralysis in mice with potency comparable to modern TeNT. Our work identifies neurotoxigenic C. tetani in ancient DNA, new Clostridium species unique to ancient human samples, and a novel variant of TeNT that can cause disease in mammals.

genomics↗

bioRxiv2021

Structure-function analysis of distant diphtheria toxin homologs reveals insights into host adaptation

Diphtheria toxin (DT) is the archetype of bacterial exotoxins implicated in human diseases and has played a central role in defining the field of toxinology since its discovery in 1888. Despite being one of the most extensively characterized bacterial toxins, the origins and molecular evolution of DT host specialization remain unknown. Here, we determined high-resolution structures of two recently discovered distant homologs of DT. These DT-like proteins from non-human associated Streptomyces albireticuli (17% identity to DT) and Seinonella peptonophila (20% identity to DT) display remarkable structural similarity to DT enabling a comparative investigation into DTs unique toxicity toward mammalian cells. We find that the individual domains of DT-like toxins retain two critical features of DTs activity: full catalytic function and ability to translocate across mammalian cell membranes. However, we show that receptor-binding, pH-dependent pore-formation and proteolytic release of the cytotoxic enzyme into the cytosol are not optimized for human cell physiology and thus unable to efficiently deliver the cytotoxic cargo into human hosts. Our work provides structural insights into DTs evolutionary history, and implies key transitions required for the emergence of human-specificity of a major bacterial exotoxin with an important history in human disease.

microbiology↗

bioRxiv2020

Telomere-to-telomere assembly of the genome of an individual Oikopleura dioica from Okinawa using Nanopore-based sequencing

BackgroundThe larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. ResultsHere, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is in five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups of OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. ConclusionsWe show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioicas highly polymorphic genome. This assembly will be a useful resource for genome-wide comparative studies between O. dioica and other species, as well as studies of chromosomal evolution in this lineage.

genomics↗

bioRxiv2020

Gene expression and in situ protein profiling of candidate SARS-CoV-2 receptors in human airway epithelial cells and lung tissue

In December 2019, SARS-CoV-2 emerged causing the COVID-19 pandemic. SARS-CoV, the agent responsible for the 2003 SARS outbreak, utilizes ACE2 and TMPRSS2 host molecules for viral entry. ACE2 and TMPRSS2 have recently been implicated in SARS-CoV-2 viral infection. Additional host molecules including ADAM17, cathepsin L, CD147, and GRP78 may also function as receptors for SARS-CoV-2. To determine the expression and in situ localization of candidate SARS-CoV-2 receptors in the respiratory mucosa, we analyzed gene expression datasets from airway epithelial cells of 515 healthy subjects, gene promoter activity analysis using the FANTOM5 dataset containing 120 distinct sample types, single cell RNA sequencing (scRNAseq) of 10 healthy subjects, immunoblots on multiple airway epithelial cell types, and immunohistochemistry on 98 human lung samples. We demonstrate absent to low ACE2 promoter activity in a variety of lung epithelial cell samples and low ACE2 gene expression in both microarray and scRNAseq datasets of epithelial cell populations. Consistent with gene expression, rare ACE2 protein expression was observed in the airway epithelium and alveoli of human lung. We present confirmatory evidence for the presence of TMPRSS2, CD147, and GRP78 protein in vitro in airway epithelial cells and confirm broad in situ protein expression of CD147 in the respiratory mucosa. Collectively, our data suggest the presence of a mechanism dynamically regulating ACE2 expression in human lung, perhaps in periods of SARS-CoV-2 infection, and also suggest that alternate receptors for SARS-CoV-2 exist to facilitate initial host cell infection.

immunology↗

bioRxiv2017

Newly identified relatives of botulinum neurotoxins shed light on their molecular evolution

The evolution of bacterial toxins is a central question to understanding the origins of human pathogens and infectious disease. Through genomic data mining, we traced the evolution of the deadliest known toxin family, clostridial neurotoxins, comprised of tetanus and botulinum neurotoxins (BoNT). We identified numerous uncharacterized lineages of BoNT-related genes in environmental species outside of Clostridium, revealing insights into their molecular ancestry. Phylogenetic analysis pinpointed a sister lineage of BoNT-like toxins in the gram-negative organism, Chryseobacterium piperi, that exhibit distant homology at the sequence level but preserve overall domain architecture. Resequencing and assembly of the C. piperi genome confirmed the presence of BoNT-like proteins encoded within two toxin-rich gene clusters. A C. piperi BoNT-like protein was validated as a novel toxin that induced necrotic cell death in human kidney cells. Mutagenesis of the putative active site abolished toxicity and indicated a zinc metalloprotease-dependent mechanism. The C. piperi toxin did not cleave common SNARE substrates of BoNTs, indicating that BoNTs have diverged from related families in substrate specificity. The new lineages of BoNT-like toxins identified by computational methods represent evolutionary missing links, and suggest an origin of clostridial neurotoxins from ancestral toxins present in environmental bacteria.\n\nSignificance statementThe origins of bacterial toxins that cause human disease is a key question in our understanding of pathogen evolution. To explore this question, we searched genomes for evolutionary relatives of the deadliest biological toxins known to science, botulinum neurotoxins. Genomic and phylogenetic analysis revealed a group of toxins in the Chryseobacterium piperi genome that are a sister lineage to botulinum toxins. Genome sequencing of this organism confirmed the presence of toxin-rich gene clusters, and a predicted C. piperi toxin was shown to induce necrotic cell death in human cells. These newly predicted toxins are missing links in our understanding of botulinum neurotoxin evolution, revealing its origins from an ancestral family of toxins that may be widespread in the environment.

bioinformatics↗