Biology subjects

Plessy, C.

Publications and source records attributed to Plessy, C..

17 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

The complete mitogenome of an unidentified Oikopleura species

1.Appendicularians are planktonic tunicates abundant all over the world. Currently, only two complete annotated mitochondrial genome assemblies are available for appendicularians, both for cryptic species of Oikopleura dioica. This underrepresentation of available appendicularian mitochondrial genomes limits environmental DNA sequencing (eDNA) studies that rely on mitochondrial markers as a taxonomic barcode. We report the complete mitochondrial genome assembly and annotation of an unknown appendicularian species isolated from the Amami Oshima island, Kagoshima prefecture, Japan, that has significant sequence difference with other currently available assemblies and will serve as a useful resource for ecological studies and further mitochondrial studies of appendicularians.

genomics↗

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

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

Automated phenol-chloroform extraction of high molecular weight genomic DNA for use in long-read single-molecule sequencing.

In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institutes Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run can be seen at http://doi.org/10.6084/m9.figshare.17097065

genomics↗

bioRxiv2021

Speed fluctuations of bacterial replisomes

Replisomes are multi-protein complexes that replicate genomes with remarkable speed and accuracy. Despite their importance, their dynamics is poorly characterized, especially in vivo. In this paper, we present an approach to infer the replisome dynamics from the DNA abundance distribution measured in a growing bacterial population. Our method is sensitive enough to detect subtle variations of the replisome speed along the genome. As an application, we experimentally measured the DNA abundance distribution in Escherichia coli populations growing at different temperatures using deep sequencing. We find that the average replisome speed increases nearly five-fold between 17{degrees}C and 37{degrees}C. Further, we observe wave-like variations of the replisome speed along the genome. These variations correlate with previously observed variations of the mutation rate. We interpret this correlation as a speed-error trade-off in DNA replication. Our approach has the potential to elucidate replication dynamics in E. coli mutants and in other bacterial species.

biophysics↗

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

Centromere-specific antibody-mediated karyotyping of Okinawan Oikopleura dioica suggests the presence of three chromosomes.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies staining properties for use in epifluorescent and confocal based fluorescent microscopy.

cell biology↗

bioRxiv2019

Widespread use of the "ascidian" mitochondrial genetic code in tunicates

BackgroundAscidians, a tunicate class, use a mitochondrial genetic code that is distinct from vertebrates and other invertebrates. Though it has been used to translate the coding sequences from other tunicate species on a case-by-case basis, it is has not been investigated whether this can be done systematically. This is an important because a) some tunicate mitochondrial sequences are currently translated with the invertebrate code by repositories such as NCBIs GenBank, and b) uncertainties about the genetic code to use can complicate or introduce errors in phylogenetic studies based on translated mitochondrial protein sequences. MethodsWe collected publicly available nucleotide sequences for non-ascidian tunicates including appendicularians such as Oikopleura dioica, translated them using the ascidian mitochondrial code, and built multiple sequence alignments covering all tunicate classes. ResultsAll tunicates studied here appear to translate AGR codons to glycine instead of serine (invertebrates) or as a stop codon (vertebrates), as initially described in ascidians. Among Oikopleuridae, we suggest further possible changes in the use of the ATA (Ile [->] Met) and TGA (Trp [->] Arg) codons. ConclusionsWe recommend using the ascidian mitochondrial code in automatic translation pipelines of mitochondrial sequences for all tunicates. Further investigation is required for additional species-specific differences.

genomics↗

bioRxiv2019

Machine-driven parameter optimisation of biochemical reactions

The development of complex, multi-step methods in molecular biology is a laborious, costly, iterative and often intuition-bound process where an optimum is sought in a multidimensional parameter space through step-by-step optimisations. The difficulty of miniaturising reactions under the microliter volumes usually handled in multiwell plates by robots, plus the cost of the experiments, limit the number of parameters and the dynamic ranges that can be explored. Nevertheless, because of non-linearities of the response of biochemical systems to their reagent concentrations, broad dynamic ranges are necessary. Here we use a high-performance nanoliter handling platform and computer generation of liquid transfer programs to explore in quadruplicates more than 600 combinations of 4 parameters of a biochemical reaction, the reverse-transcription, which lead us to uncover non-linear responses, parameter interactions and novel mechanistic insights. With the increased availability of computer-driven laboratory platforms for biotechnology, our results demonstrate the feasibility and advantage of methods development based on reproducible, computer-aided exhaustive characterisation of biochemical systems.

molecular biology↗

bioRxiv2019

RADICL-seq identifies general and cell type-specific principles of genome-wide RNA-chromatin interactions

Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding transcripts exhibit nuclear localization and several have been shown to play a role in the regulation of gene expression and chromatin remodelling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed. However, they still present some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA-chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared to existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of transcripts as well as cell type-specific RNA-chromatin interactions, and emphasizes the role of transcription in the establishment of chromatin structure.

genomics↗

bioRxiv2019

Decoding neuronal diversity by single-cell Convert-seq

The conversion of cell fates is controlled by hierarchical gene regulatory networks (GRNs) that induce remarkable alterations in cellular and transcriptome states. The identification of key regulators within these networks from myriad of candidate genes, however, poses a major research challenge. Here we present Convert-seq, combining single-cell RNA sequencing (scRNA-seq) and pooled ectopic gene expression with a new strategy to discriminate sequencing reads derived from exogenous and endogenous transcripts. We demonstrate Convert-seq by associating hundreds of single cells during reprogramming of human fibroblasts to induced neurons (iN) with exogenous and endogenous transcriptional signatures. Convert-seq identified GRNs modulating the emergence of developmental trajectories and predicted combinatorial activation of exogenous transcription factors controlling iN subtype specification. Functional validation of iN subtypes generated by novel combinations of exogenous transcription factors establish Convert-seq as a broadly applicable workflow to rapidly identify key transcription factors and GRNs orchestrating the reprogramming of virtually any cell type.

neuroscience↗

bioRxiv2018

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5-ends with an original sample multiplexing strategy in the C1 microfluidic system. We first quantified the performance of C1 CAGE and found it as accurate and sensitive as other methods in C1 system. We then used it to profile promoter and enhancer activities in the cellular response to TGF-{beta} of lung cancer cells and discovered subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand within a single-cell in a mutually exclusive manner, which was validated using single molecule fluorescence in-situ hybridization.

genomics↗

bioRxiv2017

Linking FANTOM5 CAGE Peaks To Annotations With CAGEscan

The FANTOM5 expression atlas is a quantitative measurement of the activity of nearly 200,000 promoter regions across nearly 2,000 different human primary cells, tissue types and cell lines. Generation of this atlas was made possible by the use of CAGE, an experimental approach to localise transcription start sites at single-nucleotide resolution by sequencing the 5' ends of capped RNAs after their conversion to cDNAs. While 50% of CAGE-defined promoter regions could be confidently associated to adjacent transcriptional units, nearly 100,000 promoter regions remained gene-orphan. To address this, we used the CAGEscan method, in which random-primed 5'-cDNAs are paired-end sequenced. Pairs starting in the same region are assembled in transcript models called CAGEscan clusters. Here, we present the production and quality control of CAGEscan libraries from 56 FANTOM5 RNA sources, which enhances the FANTOM5 expression atlas by providing experimental evidence associating core promoter regions with their cognate transcripts.

genomics↗

bioRxiv2016

Single-cell transcriptomes of fluorescent, ubiquitination-based cell cycle indicator cells

We used a transgenic HeLa cell line that reports cell cycle phases through fluorescent, ubiquitination-based cell cycle indicators (Fucci), to produce a reference dataset of more than 270 curated single cells. Microscopic images were taken from each cell followed by RNA-sequencing, so that single-cell expression data is associated to the fluorescence intensity of the Fucci probes in the same cell. We developed an open data management and quality control workflow that enables users to replicate the processing of the sequence and microscopic image data that we deposited in public repositories. The workflow outputs a table with metadata, that is the starting point for further studies on these data. Beyond its use for cell cycle studies, We also expect that our workflow can be adapted to other single-cell projects using a similar combination of sequencing data and fluorescence measurements.

genomics↗