Exploration des dynamiques de chromosomes par assemblage Hi-C

L'avènement des technologies de séquençage ADN à haut-debit a initié une tendance grandissante dans l'assemblage de génomes. La qualité de ces génomes est un prérequis essentiel pour comprendre les interactions au sein de et entre ces chromosomes. Nos méthodes se basent principalement sur les technologies de capture de conformation de chromosomes comme le Hi-C. Lors d'un protocole de Hi-C, les molécules d'ADN sont réticulées avec les protéines environnantes pour former un complexe protéine-ADN statique et volumineux. Ceci permet de capturer la conformation spatiale en piégeant les molécules physiquement proches dans l'espace. Ainsi, le Hi-C est très approprié pour l'analyse de la structure 3D des génomes, ce qui permet d'obtenir un certain nombre d'informations sur le génome. Il a été ainsi montré que sa structure tridimensionnelle peut être reliée directement à sa structure 1D grâce aux propriétés physiques des polymères d'ADN. De plus, une telle proximité en 3D donne également accès à des informations de compartimentation, ce qui a ouvert la voie à une nouvelle approche de binning métagénomique, connue sous le nom de meta3C. Au cours de ce travail, nous étendons ces méthodes à des études de cas présentant une complexité grandissante. Tout d'abord, nous améliorons les outils d'assemblage de génomes et démontrons leur validité avec l'assemblage de Ectocarpus sp., puis nous mettons en évidence des réarrangements chromosomiques au sein d'assemblages joints de Trichoderma reesei et Cataglyphis hispanica. Enfin, nous utilisons la même approche avec le binning métagénomique sur des échantillons de souris in vivo afin de reconstruire des centaines de génomes.The advent of high-throughput DNA sequencing technologies has set off an expanding trend in genome assembling and scaffolding. Such genome quality is an essential preliminary to understand interactions between and among chromosomes. We built upon a computational and technological framework that let us tackle genome assembly problems of increasing complexity. Our methods are mainly based on chromosome conformation capture technologies such as Hi-C. In a Hi-C experiment, DNA molecules are cross-linked with the surrounding proteins and form a large, static protein-DNA complex. This captures the spatial conformation by trapping together molecules that are physically close to each other. Therefore, Hi-C is very suitable for 3D genome structure analysis, which lets us infer a wealth of information about the genome. It was indeed shown that the tridimensional structure of the genome can be unambiguously linked to its 1D structure thanks to the physical properties of DNA polymers. Moreover, such 3D proximity also gives access to cell compartment information, thus opening the way for an additional approach for metagenomic binning, known as meta3C. In this work, we expand upon these methods and apply them to use cases with more and more complexity. We first improve on tools for genome assembly and demonstrate their validity with the scaffolding of Ectocarpus sp., then unveil rearrangements in joint scaffoldings of Trichoderma reesei and Cataglyphis hispanica. Lastly, we use the same approach with metagenomic binning on live mouse microbiome samples to reconstruct hundreds of genomes

Exploration des dynamiques de chromosomes par assemblage Hi-C

The advent of high-throughput DNA sequencing technologies has set off an expanding trend in genome assembling and scaffolding. Such genome quality is an essential preliminary to understand interactions between and among chromosomes. We built upon a computational and technological framework that let us tackle genome assembly problems of increasing complexity. Our methods are mainly based on chromosome conformation capture technologies such as Hi-C. In a Hi-C experiment, DNA molecules are cross-linked with the surrounding proteins and form a large, static protein-DNA complex. This captures the spatial conformation by trapping together molecules that are physically close to each other. Therefore, Hi-C is very suitable for 3D genome structure analysis, which lets us infer a wealth of information about the genome. It was indeed shown that the tridimensional structure of the genome can be unambiguously linked to its 1D structure thanks to the physical properties of DNA polymers. Moreover, such 3D proximity also gives access to cell compartment information, thus opening the way for an additional approach for metagenomic binning, known as meta3C. In this work, we expand upon these methods and apply them to use cases with more and more complexity. We first improve on tools for genome assembly and demonstrate their validity with the scaffolding of Ectocarpus sp., then unveil rearrangements in joint scaffoldings of Trichoderma reesei and Cataglyphis hispanica. Lastly, we use the same approach with metagenomic binning on live mouse microbiome samples to reconstruct hundreds of genomes.L'avènement des technologies de séquençage ADN à haut-debit a initié une tendance grandissante dans l'assemblage de génomes. La qualité de ces génomes est un prérequis essentiel pour comprendre les interactions au sein de et entre ces chromosomes. Nos méthodes se basent principalement sur les technologies de capture de conformation de chromosomes comme le Hi-C. Lors d'un protocole de Hi-C, les molécules d'ADN sont réticulées avec les protéines environnantes pour former un complexe protéine-ADN statique et volumineux. Ceci permet de capturer la conformation spatiale en piégeant les molécules physiquement proches dans l'espace. Ainsi, le Hi-C est très approprié pour l'analyse de la structure 3D des génomes, ce qui permet d'obtenir un certain nombre d'informations sur le génome. Il a été ainsi montré que sa structure tridimensionnelle peut être reliée directement à sa structure 1D grâce aux propriétés physiques des polymères d'ADN. De plus, une telle proximité en 3D donne également accès à des informations de compartimentation, ce qui a ouvert la voie à une nouvelle approche de binning métagénomique, connue sous le nom de meta3C. Au cours de ce travail, nous étendons ces méthodes à des études de cas présentant une complexité grandissante. Tout d'abord, nous améliorons les outils d'assemblage de génomes et démontrons leur validité avec l'assemblage de Ectocarpus sp., puis nous mettons en évidence des réarrangements chromosomiques au sein d'assemblages joints de Trichoderma reesei et Cataglyphis hispanica. Enfin, nous utilisons la même approche avec le binning métagénomique sur des échantillons de souris in vivo afin de reconstruire des centaines de génomes

Scaffolding bacterial genomes and probing host-virus interactions in gut microbiome by proximity ligation (chromosome capture) assay

International audienceThe biochemical activities of microbial communities, or microbiomes, are essential parts of environmental and animal ecosystems. The dynamics, balance, and effects of these communities are strongly influenced by phages present in the population. Being able to characterize bacterium-phage relationships is therefore essential to investigate these ecosystems to the full extent of their complexity. However, this task is currently limited by (i) the ability to characterize complete bacterial and viral genomes from a complex mix of species and (ii) the difficulty to assign phage sequences to their bacterial hosts. We show that both limitations can be circumvented using meta3C, an experimental and computational approach that exploits the physical contacts between DNA molecules to infer their proximity. In a single experiment, dozens of bacterial and phage genomes present in a complex mouse gut microbiota were assembled and scaffolded de novo. The phage genomes were then assigned to their putative bacterial hosts according to the physical contacts between the different DNA molecules, opening new perspectives for a comprehensive picture of the genomic structure of the gut flora. Therefore, this work holds far-reaching implications for human health studies aiming to bridge the virome to the microbiome

Serpentine: a flexible 2D binning method for differential Hi-C analysis

International audienceHi-C contact maps reflect the relative contact frequencies between pairs of genomic loci, quantified through deep sequencing. Differential analyses of these maps enable downstream biological interpretations. However, the multi-fractal nature of the chromatin polymer inside the cellular envelope results in contact frequency values spanning several orders of magnitude: contacts between loci pairs at large genomic distances are much sparser than closer pairs. The same is true for poorly-covered regions such as repeated sequences. Both distant and poorly covered regions translate into low signal-to-noise ratios. There is no clear consensus to address this limitation

MetaTOR: A Computational Pipeline to Recover High-Quality Metagenomic Bins From Mammalian Gut Proximity-Ligation (meta3C) Libraries

International audienceCharacterizing the complete genomic structure of complex microbial communities would represent a key step toward the understanding of their diversity, dynamics, and evolution. Current metagenomics approaches aiming at this goal are typically done by analyzing millions of short DNA sequences directly extracted from the environment. New experimental and computational approaches are constantly sought for to improve the analysis and interpretation of such data. We developed MetaTOR, an open-source computational solution that bins DNA contigs into individual genomes according to their 3D contact frequencies. Those contacts are quantified by chromosome conformation capture experiments (3C, Hi-C), also known as proximity-ligation approaches, applied to metagenomics samples (meta3C). MetaTOR was applied on 20 meta3C libraries of mice gut microbiota. We quantified the program ability to recover high-quality metagenome-assembled genomes (MAGs) from metagenomic assemblies generated directly from the meta3C libraries. Whereas nine high-quality MAGs are identified in the 148-Mb assembly generated using a single meta3C library, MetaTOR identifies 82 high-quality MAGs in the 763-Mb assembly generated from the merged 20 meta3C libraries, corresponding to nearly a third of the total assembly. Compared to the hybrid binning softwares MetaBAT or CONCOCT, MetaTOR recovered three times more high-quality MAGs. These results underline the potential of 3C-/Hi-C-based approaches in metagenomic projects

Normalization of Chromosome Contact Maps: Matrix Balancing and Visualization

International audienceOver the last decade, genomic proximity ligation approaches have reshaped our vision of chromosomes 3D organizations, from bacteria nucleoids to larger eukaryotic genomes. The different protocols (3Cseq, Hi-C, TCC, MicroC [XL], Hi-CO, etc.) rely on common steps (chemical fixation digestion, ligation…) to detect pairs of genomic positions in close proximity. The most common way to represent these data is a matrix, or contact map, which allows visualizing the different chromatin structures (compartments, loops, etc.) that can be associated to other signals such as transcription, protein occupancy, etc. as well as, in some instances, to biological functions.In this chapter we present and discuss the filtering of the events recovered in proximity ligation experiments as well as the application of the balancing normalization procedure on the resulting contact map. We also describe a computational tool for visualizing normalized contact data dubbed Scalogram.The different processes described here are illustrated and supported by the laboratory custom-made scripts pooled into "hicstuff," an open-access python package accessible on github ( https://github.com/koszullab/hicstuff )

Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes

International audienceBackground: The presence of low complexity and repeated regions in genomes often results in difficulties to assemblesequencing data into full chromosomes. However, the availability of full genome scaffolds is essential to severalinvestigations, regarding for instance the evolution of entire clades, the analysis of chromosome rearrangements,and is pivotal to sexual crossing studies. In non-conventional but industrially relevant model organisms, such as theascomycete Trichoderma reesei, a complete genome assembly is seldom available.Results: The chromosome scaffolds of T. reesei QM6a and Rut-C30 strains have been generated using a contactgenomic/proximity ligation genomic approach. The original reference assembly, encompassing dozens of scaffolds,was reorganized into two sets of seven chromosomes. Chromosomal contact data also allowed to characterize10–40 kb, gene-free, AT-rich (76%) regions corresponding to the T. reesei centromeres. Large chromosomal rearrangements(LCR) in Rut-C30 were then characterized, in agreement with former studies, and the position of LCRbreakpoints used to assess the likely chromosome structure of other T. reesei strains [QM9414, CBS999.97 (1-1, re), andQM9978]. In agreement with published results, we predict that the numerous chromosome rearrangements found inhighly mutated industrial strains may limit the efficiency of sexual reproduction for their improvement.Conclusions: The GRAAL program allowed us to generate the karyotype of the Rut-C30 strain, and from there topredict chromosome structure for most T. reesei strains for which sequence is available. This method that exploitsproximity ligation sequencing approach is a fast, cheap, and straightforward way to characterize both chromosomestructure and centromere sequences and is likely to represent a popular convenient alternative to expensive andwork-intensive resequencing projects

MOESM2 of Proximity ligation scaffolding and comparison of two Trichoderma reesei strains genomes

Additional file 2. QM6a reassembly sequence in fasta format (7 chromosomes + unassembled scaffolds)