Genomics in the long-read sequencing era van Dijk, Erwin L.; Naquin, Delphine; Gorrichon, Kévin ...
Trends in genetics,
09/2023, Letnik:
39, Številka:
9
Journal Article
Recenzirano
Long-read sequencing (LRS) methods can now produce highly accurate (ultra)long reads and thus enable for the first time the production of truly complete telomere-to-telomere (T2T) assemblies of ...complex genomes.While the recently produced T2T assemblies were labor-intensive and required a combination of various techniques, in the near future T2T assemblies based on LRS alone will be produced.We have entered the era of population scale long-read sequencing, where graph-based pangenomes much better representing genomic variation will increasingly be used in the near future.New applications of LRS appear at a rapid pace, with examples ranging from higher order chromatin interaction studies to the quality control of mRNA vaccines.
Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.
Abstract
Bacteria of the genus
Streptomyces
are prolific producers of specialized metabolites, including antibiotics. The linear chromosome includes a central region harboring core genes, as well as ...extremities enriched in specialized metabolite biosynthetic gene clusters. Here, we show that chromosome structure in
Streptomyces ambofaciens
correlates with genetic compartmentalization during exponential phase. Conserved, large and highly transcribed genes form boundaries that segment the central part of the chromosome into domains, whereas the terminal ends tend to be transcriptionally quiescent compartments with different structural features. The onset of metabolic differentiation is accompanied by a rearrangement of chromosome architecture, from a rather ‘open’ to a ‘closed’ conformation, in which highly expressed specialized metabolite biosynthetic genes form new boundaries. Thus, our results indicate that the linear chromosome of
S. ambofaciens
is partitioned into structurally distinct entities, suggesting a link between chromosome folding, gene expression and genome evolution.
Abstract Centrioles are the core constituent of centrosomes, microtubule-organizing centers involved in directing mitotic spindle assembly and chromosome segregation in animal cells. In sexually ...reproducing species, centrioles degenerate during oogenesis and female meiosis is usually acentrosomal. Centrioles are retained during male meiosis and, in most species, are reintroduced with the sperm during fertilization, restoring centriole numbers in embryos. In contrast, the presence, origin, and function of centrioles in parthenogenetic species is unknown. We found that centrioles are maternally inherited in two species of asexual parthenogenetic nematodes and identified two different strategies for maternal inheritance evolved in the two species. In Rhabditophanes diutinus , centrioles organize the poles of the meiotic spindle and are inherited by both the polar body and embryo. In Disploscapter pachys , the two pairs of centrioles remain close together and are inherited by the embryo only. Our results suggest that maternally-inherited centrioles organize the embryonic spindle poles and act as a symmetry-breaking cue to induce embryo polarization. Thus, in these parthenogenetic nematodes, centrioles are maternally-inherited and functionally replace their sperm-inherited counterparts in sexually reproducing species.
causes severe foodborne illness in pregnant women and immunocompromised individuals. After the intestinal phase of infection, the liver plays a central role in the clearance of this pathogen through ...its important functions in immunity. However, recent evidence suggests that during long-term infection of hepatocytes, a subpopulation of
may escape eradication by entering a persistence phase in intracellular vacuoles. Here, we examine whether this long-term infection alters hepatocyte defense pathways, which may be instrumental for bacterial persistence. We first optimized cell models of persistent infection in human hepatocyte cell lines HepG2 and Huh7 and primary mouse hepatocytes (PMH). In these cells,
efficiently entered the persistence phase after three days of infection, while inducing a potent interferon response, of type I in PMH and type III in HepG2, while Huh7 remained unresponsive. RNA-sequencing analysis identified a common signature of long-term
infection characterized by the overexpression of a set of genes involved in antiviral immunity and the under-expression of many acute phase protein (APP) genes, particularly involved in the complement and coagulation systems. Infection also altered the expression of cholesterol metabolism-associated genes in HepG2 and Huh7 cells. The decrease in APP transcripts was correlated with lower protein abundance in the secretome of infected cells, as shown by proteomics, and also occurred in the presence of APP inducers (IL-6 or IL-1β). Collectively, these results reveal that long-term infection with
profoundly deregulates the innate immune functions of hepatocytes, which could generate an environment favorable to the establishment of persistent infection.
With its nuclear dualism, the ciliate
constitutes a unique model to study how host genomes cope with transposable elements (TEs).
harbors two germline micronuclei (MICs) and a polyploid somatic ...macronucleus (MAC) that develops from one MIC at each sexual cycle. Throughout evolution, the MIC genome has been continuously colonized by TEs and related sequences that are removed from the somatic genome during MAC development. Whereas TE elimination is generally imprecise, excision of approximately 45,000 TE-derived internal eliminated sequences (IESs) is precise, allowing for functional gene assembly. Programmed DNA elimination is concomitant with genome amplification. It is guided by noncoding RNAs and repressive chromatin marks. A subset of IESs is excised independently of this epigenetic control, raising the question of how IESs are targeted for elimination. To gain insight into the determinants of IES excision, we established the developmental timing of DNA elimination genome-wide by combining fluorescence-assisted nuclear sorting with high-throughput sequencing. Essentially all IESs are excised within only one endoreplication round (32C to 64C), whereas TEs are eliminated at a later stage. We show that DNA elimination proceeds independently of replication. We defined four IES classes according to excision timing. The earliest excised IESs tend to be independent of epigenetic factors, display strong sequence signals at their ends, and originate from the most ancient integration events. We conclude that old IESs have been optimized during evolution for early and accurate excision by acquiring stronger sequence determinants and escaping epigenetic control.
Dendritic cell-derived exosomes (Dex) are nanovesicles bearing major histocompatibility complexes promoting T-cell-dependent antitumor effects in mice. Two phase I clinical trials aimed at ...vaccinating cancer patients with peptide-pulsed Dex have shown the feasibility and safety of inoculating clinical-grade Dex, but have failed to show their immunizing capacity. These low immunogenic capacities have led us to develop second-generation Dex with enhanced immunostimulatory properties. Here, we show that interferon-γ is a key cytokine conditioning the dendritic cell to induce the expression of CD40, CD80, CD86, and CD54 on Dex, endowing them with direct and potent peptide-dependent CD8(+) T-cell-triggering potential in vitro and in vivo. In this study, we describe the clinical grade process to manufacture large-scale interferon-γ-Dex vaccines and their quality control parameters currently used in a phase II trial.
Abstract
Cinnamyl alcohol dehydrogenase is one of the enzymes controlling the first two committed steps of lignification. Using a 3-dimensional similarity model of this enzyme, a series of novel ...phos-phonates (1-5) was designed as potential inhibitors. Phosphonates 1-5 were synthesized in good yield by reaction of the corresponding cinnamaldehydes with tetraethylmethylene diphos-phonate. Monophosphonic acids 6 and 7 were obtained by basic hydrolysis of the corresponding phosphonates while phosphonamidate 8 was synthesized by reacting benzylamine with the iminium salt intermediate of the monophosphonic acid. Using recombinant cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) the inhibitory activity of these compounds was evaluated and compared with that of the carbonyl analogues. Inhibition kinetic studies showed compounds 2 and 3 to be mixed type linear inhibitors while compound 4 was uncompetitive. H NMR studies of inhibitor 2, for which K1 and K1 were 20 and 86 pM, respectively, in the presence of CAD based on selective line-broadening showed an increased interaction of the 3-Ome group of the aromatic ring of the inhibitor with the active site of the CAD. A transferred nuclear overhauser effect spectroscopy (TRNOESY) experiment for inhibitor 2 with CAD was used to determine the conformation of this compound bound to CAD. These results were found to be consistent with the 3-dimensional structural model of the enzyme
PDF
