Micro-C Analysis Workflow Using Pairtools and Juicer.

Three-dimensional (3D) chromosome structures are closely related to various chromosomal functions, and deep analysis of the structures is crucial for the elucidation of the functions. In recent years, chromosome conformation capture (3C) techniques combined with next-generation sequencing analysis have been developed to comprehensively reveal 3D chromosome structures. Micro-C is one such method that can detect the structures at nucleosome resolution. In this chapter, I provide a basic method for Micro-C analysis. I present and discuss a series of data analyses ranging from mapping to basic downstream analyses, including loop detection.

Read Mapping for Hi-C Analysis.

Hi-C is a popular ligation-based technique to detect 3D physical chromosome structure within the nucleus using cross-linking and next-generation sequencing. As an unbiased genome-wide assay based on chromosome conformation capture, it provides rich insights into chromosome structure, dynamic chromosome folding and interactions, and the regulatory state of a cell. Bioinformatics analyses of Hi-C data require dedicated protocols as most genome alignment tools assume that both paired-end reads will map to the same chromosome, resulting in large two-dimensional matrices as processed data. Here, we outline the necessary steps to generate high-quality aligned Hi-C data by separately mapping each read while correcting for biases from restriction enzyme digests. We introduce our own custom open-source pipeline, which enables users to select an aligner of their choosing with high accuracy and performance. This enables users to generate high-resolution datasets with fast turnaround and fewer unmapped reads. Finally, we discuss recent innovations in experimental techniques, bioinformatics techniques, and their applications in clinical testing for diagnostics.

Reconstruction of 3D Chromosome Structure from Single-Cell Hi-C Data via Recurrence Plots.

We describe an approach for reconstructing three-dimensional (3D) structures from single-cell Hi-C data. This approach has been inspired by a method of recurrence plots and visualization tools for nonlinear time series data. Some examples are also presented.

Extracting Chromosome Structural Information as One-Dimensional Metrics and Integrating Them with Epigenomics.

Hi-C is a powerful method for obtaining genome-wide chromosomal structural information. The typical Hi-C analysis utilizes a two-dimensional (2D) contact matrix, which poses challenges for quantitative comparisons, visualizations, and integrations across multiple datasets. Here, we present a protocol for extracting one-dimensional (1D) features from chromosome structure data by HiC1Dmetrics. Leveraging these 1D features enables integrated analysis of Hi-C and epigenomic data.

Selecting reference genes for RT-qPCR studies involving the presence of B chromosomes in Psalidodon (Characiformes, Characidae).

BACKGROUND: B chromosomes are extra non-essential elements present in several eukaryotes. Unlike A chromosomes which are essential and present in all individuals of a species, B chromosomes are not necessary for normal functioning of an organism. Formerly regarded as genetically inactive, B chromosomes have been discovered to not only express their own genes, but also to exert influence on gene expression in A chromosomes. Recent studies have shown that, in some Psalidodon (Characiformes, Characidae) species, B chromosomes might be associated with phenotypic effects, such as changes in the reproductive cycle and gene expression. METHODS AND RESULTS: In this study, we aimed to establish stable reference genes for RT-qPCR experiments conducted on gonads of three fish species within Psalidodon genus, both in the presence and absence of B chromosomes. The stability of five selected reference genes was assessed using NormFinder, geNorm, BestKeeper, and RefFinder algorithms. We determined ppiaa and pgk1 as the most stable genes in P. fasciatus, whereas ppiaa and hmbsa showed the highest stability in P. bockmanni. For P. paranae, tbp and hprt1 were the most stable genes in females, and ppiaa and hprt1 were the most stable in males. CONCLUSIONS: We determined the most stable reference genes in gonads of three Psalidodon species considering the presence of B chromosomes. This is the first report of reference gene stability in the genus and provides valuable tools to better understand the effects of B chromosomes at gene expression level.

Genomes were 'scrambled' when worms left the sea.

Chromosomal chaos may have aided their moves to fresh water and land.

Chromosome-level genome and population genomics of the intermediate horseshoe bat ( Rhinolophus affinis) reveal the molecular basis of virus tolerance in Rhinolophus and echolocation call frequency variation.

Horseshoe bats (genus Rhinolophus, family Rhinolophidae) represent an important group within chiropteran phylogeny due to their distinctive traits, including constant high-frequency echolocation, rapid karyotype evolution, and unique immune system. Advances in evolutionary biology, supported by high-quality reference genomes and comprehensive whole-genome data, have significantly enhanced our understanding of species origins, speciation mechanisms, adaptive evolutionary processes, and phenotypic diversity. However, genomic research and understanding of the evolutionary patterns of Rhinolophus are severely constrained by limited data, with only a single published genome of R. ferrumequinum currently available. In this study, we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat ( R. affinis). Comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae. Notably, we observed expansions in several immune-related gene families and identified various genes functionally associated with the SARS-CoV-2 signaling pathway, DNA repair, and apoptosis, which displayed signs of rapid evolution. In addition, we observed an expansion of the major histocompatibility complex class II (MHC-II) region and a higher copy number of the HLA- DQB2 gene in horseshoe bats compared to other chiropteran species. Based on whole-genome resequencing and population genomic analyses, we identified multiple candidate loci (e.g., GLI3) associated with variations in echolocation call frequency across R. affinis subspecies. This research not only expands our understanding of the genetic characteristics of the Rhinolophus genus but also establishes a valuable foundation for future research.

Spectral-based detection of chromatin loops in multiplexed super-resolution FISH data.

Involved in mitotic condensation, interaction of transcriptional regulatory elements and isolation of structural domains, loop formation has become a paradigm in the deciphering of chromatin architecture and its functional role. Despite the emergence of increasingly powerful genome visualization techniques, the high variability in cell populations and the randomness of conformations still make loop detection a challenge. We introduce an approach for determining the presence and frequency of loops in a collection of experimental conformations obtained by multiplexed super-resolution imaging. Based on a spectral approach, in conjunction with neural networks, this method offers a powerful tool to detect loops in large experimental data sets, both at the population and single-cell levels. The method's performance is confirmed on experimental FISH data where Hi-C and other loop detection results are available. The method is then applied to recently published experimental data, where it provides a detailed and statistically quantified description of the global architecture of the chromosomal region under study.

The chromolinker hypothesis: Are eukaryotic genomes also circular?

Over more than the past century, reports that chromosomes in Eukaryotes are linked have been published. Recently this has been confirmed by micromanipulation. The chromolinkers are DNAse sensitive, as has been previously reported. The arguments for and against chromolinkers have been reviewed, and a call for definitive research made, because if chromolinkers do exist, the whole basis for genetics may require revision.

The mitochondrial genome of Bottapotamon fukienense (Brachiura: Potamidae) is fragmented into two chromosomes.

BACKGROUND: China is the hotspot of global freshwater crab diversity, but their wild populations are facing severe pressures associated with anthropogenic factors, necessitating the need to map their taxonomic and genetic diversity and design conservation policies. RESULTS: Herein, we sequenced the mitochondrial genome of a Chinese freshwater crab species Bottapotamon fukienense, and found that it is fragmented into two chromosomes. We confirmed that fragmentation was not limited to a single specimen or population. Chromosome 1 comprised 15,111 base pairs (bp) and there were 26 genes and one pseudogene (pseudo-nad1) encoded on it. Chromosome 2 comprised 8,173 bp and there were 12 genes and two pseudogenes (pseudo-trnL2 and pseudo-rrnL) encoded on it. Combined, they comprise the largest mitogenome (23,284 bp) among the Potamidae. Bottapotamon was the only genus in the Potamidae dataset exhibiting rearrangements of protein-coding genes. Bottapotamon fukienense exhibited average rates of sequence evolution in the dataset and did not differ in selection pressures from the remaining Potamidae. CONCLUSIONS: This is the first experimentally confirmed fragmentation of a mitogenome in crustaceans. While the mitogenome of B. fukienense exhibited multiple signs of elevated mitogenomic architecture evolution rates, including the exceptionally large size, duplicated genes, pseudogenisation, rearrangements of protein-coding genes, and fragmentation, there is no evidence that this is matched by elevated sequence evolutionary rates or changes in selection pressures.

Chromosome-level genome assembly of the glass catfish ( Kryptopterus vitreolus) reveals molecular clues to its transparent phenotype.

Glass catfish ( Kryptopterus vitreolus) are notable in the aquarium trade for their highly transparent body pattern. This transparency is due to the loss of most reflective iridophores and light-absorbing melanophores in the main body, although certain black and silver pigments remain in the face and head. To date, however, the molecular mechanisms underlying this transparent phenotype remain largely unknown. To explore the genetic basis of this transparency, we constructed a chromosome-level haplotypic genome assembly for the glass catfish, encompassing 32 chromosomes and 23 344 protein-coding genes, using PacBio and Hi-C sequencing technologies and standard assembly and annotation pipelines. Analysis revealed a premature stop codon in the putative albinism-related tyrp1b gene, encoding tyrosinase-related protein 1, rendering it a nonfunctional pseudogene. Notably, a synteny comparison with over 30 other fish species identified the loss of the endothelin-3 ( edn3b) gene in the glass catfish genome. To investigate the role of edn3b, we generated edn3b -/- mutant zebrafish, which exhibited a remarkable reduction in black pigments in body surface stripes compared to wild-type zebrafish. These findings indicate that edn3b loss contributes to the transparent phenotype of the glass catfish. Our high-quality chromosome-scale genome assembly and identification of key genes provide important molecular insights into the transparent phenotype of glass catfish. These findings not only enhance our understanding of the molecular mechanisms underlying transparency in glass catfish, but also offer a valuable genetic resource for further research on pigmentation in various animal species.

Visualization and Quantification of Rapid Chromosome Movements at Early Stages of Mouse Meiosis.

Telomere-led rapid chromosome movements (RPMs) are a conserved characteristic of chromosome dynamics in meiosis. RPMs have been suggested to influence critical meiotic functions such as DNA repair and the association of the homologous chromosomes. Here, we describe a method using 3D time-lapse fluorescence imaging to monitor RPMs in Hoechst-stained mouse seminiferous tubules explants. We supplement visualization with customized quantitative motion analysis and in silico simulation. The ability to carry out live imaging, combined with quantitative image analysis, offers a sensitive tool to investigate the regulation of RPMs, chromosome reorganizations that precede dynamic mid-prophase events, and their contribution to faithful transmission of genetic information.

Effect of active loop extrusion on the two-contact correlations in the interphase chromosome.

The population-averaged contact maps generated by the chromosome conformation capture technique provide important information about the average frequency of contact between pairs of chromatin loci as a function of the genetic distance between them. However, these datasets do not tell us anything about the joint statistics of simultaneous contacts between genomic loci in individual cells. This kind of statistical information can be extracted using the single-cell Hi-C method, which is capable of detecting a large fraction of simultaneous contacts within a single cell, as well as through modern methods of fluorescent labeling and super-resolution imaging. Motivated by the prospect of the imminent availability of relevant experimental data, in this work, we theoretically model the joint statistics of pairs of contacts located along a line perpendicular to the main diagonal of the single-cell contact map. The analysis is performed within the framework of an ideal polymer model with quenched disorder of random loops, which, as previous studies have shown, allows us to take into account the influence of the loop extrusion process on the conformational properties of interphase chromatin.

Comparative karyotypic study of fifteen cyprinids (Cyprinidae, Cyprininae) species. An insight into the chromosomal evolution of the tribe Systomini.

The family Cyprinidae is the largest freshwater fish group with 377 genera and over 3,000 described species. However, this group of fish has very limited cytogenetics and advanced molecular cytogenetics information. Therefore, in this study the karyotypes and other chromosomal characteristics of 15 species in the tribe Systomini (Cyprininae) were examined using Ag-NOR staining along with fluorescence in situ hybridization (5S and 18S rDNA). All species share a similar karyotype (2n = 50; NF = 88-100) in both sexes and no differentiated sex chromosome was observed. Chromosomes bearing NOR sites ranged from one to four pairs among the species, mostly mapped adjacent to telomeres in the short arms of distinct pairs in all analyzed species. This difference indicates an extensive rearrangement of chromosomes including genomic differences. The use of the 5S and 18S rDNA probe confirmed the Ag-NOR sites interstitially located in the telomeric regions of distinct chromosomes, characterizing an interspecies variation of these sites. In most of its analyzed species, the signals of 18S rDNA probe corresponded to the Ag-NOR regions, except in Barbonymus altus, B. gonionotus, B. schwanenfeldii and Puntius brevis having these signals on the same as Ag-NOR regions and other sites.

Modular Assembly of Synthetic Secondary Chromosomes.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.

Chromosome-Scale Genome Assembly of the Sheep-Biting Louse Bovicola ovis Using Nanopore Sequencing Data and Pore-C Analysis.

Bovicola ovis, commonly known as the sheep-biting louse, is an ectoparasite that adversely affects the sheep industry. Sheep louse infestation lowers the quality of products, including wool and leather, causing a loss of approximately AUD 123M per annum in Australia alone. The lack of a high-quality genome assembly for the sheep-biting louse, as well as any closely related livestock lice, has hindered the development of louse research and management control tools. In this study, we present the assembly of B. ovis with a genome size of ~123 Mbp based on a nanopore long-read sequencing library and Illumina RNA sequencing, complemented with a chromosome-level scaffolding using the Pore-C multiway chromatin contact dataset. Combining multiple alignment and gene prediction tools, a comprehensive annotation on the assembled B. ovis genome was conducted and recalled 11,810 genes as well as other genomic features including orf, ssr, rRNA and tRNA. A manual curation using alignment with the available closely related louse species, Pediculus humanus, increased the number of annotated genes to 16,024. Overall, this study reported critical genetic resources and biological insights for the advancement of sheep louse research and the development of sustainable control strategies in the sheep industry.

Puzzle Hi-C: An accurate scaffolding software.

High-quality, chromosome-scale genomes are essential for genomic analyses. Analyses, including 3D genomics, epigenetics, and comparative genomics rely on a high-quality genome assembly, which is often accomplished with the assistance of Hi-C data. Curation of genomes reveal that current Hi-C-assisted scaffolding algorithms either generate ordering and orientation errors or fail to assemble high-quality chromosome-level scaffolds. Here, we offer the software Puzzle Hi-C, which uses Hi-C reads to accurately assign contigs or scaffolds to chromosomes. Puzzle Hi-C uses the triangle region instead of the square region to count interactions in a Hi-C heatmap. This strategy dramatically diminishes scaffolding interference caused by long-range interactions. This software also introduces a dynamic, triangle window strategy during assembly. Initially small, the window expands with interactions to produce more effective clustering. Puzzle Hi-C outperforms available scaffolding tools.

A chromosome-level genome assembly and evolutionary analysis of Coregonus ussuriensis Berg.

Coregonus ussuriensis Berg, distributed widely in cold waters above 45° N latitude, is a savored freshwater whitefish that has been included in the list of endangered animals as a consequence of overfishing. Lack of genomic information seriously hampers evolutionary and genetic research on C. ussuriensis warranting the need to assemble a high-quality reference genome to promote its genetic breeding. We assembled and constructed a reference chromosome-level C. ussuriensis genome (sequence length, 2.51 Gb; contig N50 length, 4.27 Mb) using PacBio sequencing and Hi-C assembly technology, 3,109 contigs were assembled into scaffolds, resulting in a genome assembly with 40 chromosomes and a scaffold N50 length of 62.20 Mb. In addition, 43,320 protein-coding genes were annotated. The peak Ks position in the species comparison reflects the whole-genome replication event of C. ussuriensis. This chromosome-level genome provides reference data for further studies on the molecular breeding of C. ussuriensis.

Formation and resolution of meiotic chromosome entanglements and interlocks.

Interactions between parental chromosomes during the formation of gametes can lead to entanglements, entrapments and interlocks between unrelated chromosomes. If unresolved, these topological constraints can lead to misregulation of exchanges between chromosomes and to chromosome mis-segregation. Interestingly, these configurations are largely resolved by the time parental chromosomes are aligned during pachytene. In this Review, we highlight the inevitability of topologically complex configurations and discuss possible mechanisms to resolve them. We focus on the dynamic nature of a conserved chromosomal interface - the synaptonemal complex - and the chromosome movements that accompany meiosis as potential mechanisms to resolve topological constraints. We highlight the advantages of the nematode Caenorhabditis elegans for understanding biophysical features of the chromosome axis and synaptonemal complex that could contribute to mechanisms underlying interlock resolution. In addition, we highlight advantages of using the zebrafish, Danio rerio, as a model to understand how entanglements and interlocks are avoided and resolved.