High-resolution Chromosome Research Articles

PneumoBrowse 2: an integrated visual platform for curated genome annotation and multiomics data analysis of Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic human pathogen responsible for high morbidity and mortality rates. Extensive genome sequencing revealed its large pangenome, serotype diversity, and provided insight into genome dynamics. However, functional genome analysis has lagged behind, as that requires detailed and time-consuming manual curation of genome annotations and integration of genomic and phenotypic data. To remedy this, PneumoBrowse was presented in 2018, a user-friendly interactive online platform, which provided the detailed annotation of the S. pneumoniae D39V genome, alongside transcriptomic data. Since 2018, many new studies on S. pneumoniae genome biology and protein functioning have been performed. Here, we present PneumoBrowse 2 (https://veeninglab.com/pneumobrowse), fully rebuilt in JBrowse 2. We updated annotations for transcribed and transcriptional regulatory features in the D39V genome. We added genome-wide data tracks for high-resolution chromosome conformation capture (Hi-C) data, chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq), ribosome profiling, CRISPRi-seq gene essentiality data and more. Additionally, we included 18 phylogenetically diverse S. pneumoniae genomes and their annotations. By providing easy access to diverse high-quality genome annotations and links to other databases (including UniProt and AlphaFold), PneumoBrowse 2 will further accelerate research and development into preventive and treatment strategies, through increased understanding of the pneumococcal genome.

A chromosome-level genome assembly of a deep-sea symbiotic Aplacophora mollusc Chaetoderma sp.

The worm-shaped, shell-less Caudofoveata is one of the least known groups of molluscs. As early-branching molluscs, the lack of high-quality genomes hinders our understanding of their evolution and ecology. Here, we report a high-quality chromosome-scale genome of Chaetoderma sp. combining PacBio, Illumina, and high-resolution chromosome conformation capture sequencing. The final assembly has a size of 2.45 Gb, with a scaffold N50 length of 141.46 Mb, and is anchored to 17 chromosomes. Gene annotations showed a high level of accuracy and completeness, with 23,675 predicted protein-coding genes and 94.44% of the metazoan conserved genes by BUSCO assessment. We further present 16S rRNA gene amplicon sequencing of the gut microbiota in Chaetoderma sp., which was dominated by the chemoautotrophic bacteria (phylum Gammaproteobacteria). This chromosome-level genome assembly presents the first genome for the Caudofoveata, which constitutes an important resource for studies ranging from molluscan evolution, symposium, to deep-sea adaptation.

Chromosome-level genome assembly and population genomic analysis reveal evolution and local adaptation in common hairfin anchovy (Setipinna tenuifilis).

Understanding the genetic structure and the factors associated with adaptive diversity has significant implications for the effective management of wild populations under threat from overfishing and climate change. The common hairfin anchovy (Setipinna tenuifilis) is an economically and ecologically important pelagic fish species, spanning a broad latitudinal gradient along marginal seas of the Northwest Pacific. In this study, we constructed the first reference genome of S. tenuifilis using PacBio long reads and high-resolution chromosome conformation capture (Hi-C) technology. The assembled genome was 798.38 Mb with a contig N50 of 1.43 Mb and a scaffold N50 of 32.42 Mb, which were anchored onto 24 pseudochromosomes. A total of 22,019 genes were functionally annotated, which accounted for 95.27% of the predicted protein-coding genes. Chromosomal collinearity analysis revealed chromosome fusion or fission events in Clupeiformes species. Three genetic groups of S. tenuifilis were revealed along the Chinese coast using restriction site-associated DNA sequencing (RADseq). We investigated the influence of four bioclimatic variables as potential drivers of adaptive divergence in S. tenuifilis, suggesting that these environmental variables, especially sea surface temperature, may play important roles as drivers of spatially varying selection for S. tenuifilis. We also identified candidate functional genes underlying adaptive mechanisms and ecological tradeoffs using redundancy analysis (RDA) and BayeScan analysis. In summary, this study sheds light on the evolution and spatial patterns of genetic variation of S. tenuifilis, providing a valuable genomic resource for further biological and genetic studies on this species and other closely related Clupeiformes.

The Mediator complex regulates enhancer-promoter interactions

Enhancer-mediated gene activation generally requires physical proximity between enhancers and their target gene promoters. However, the molecular mechanisms by which interactions between enhancers and promoters are formed are not well understood. Here, we investigate the function of the Mediator complex in the regulation of enhancer-promoter interactions, by combining rapid protein depletion and high-resolution MNase-based chromosome conformation capture approaches. We show that depletion of Mediator leads to reduced enhancer-promoter interaction frequencies, which are associated with a strong decrease in gene expression. In addition, we find increased interactions between CTCF-binding sites upon Mediator depletion. These changes in chromatin architecture are associated with a redistribution of the Cohesin complex on chromatin and a reduction in Cohesin occupancy at enhancers. Together, our results indicate that the Mediator and Cohesin complexes contribute to enhancer-promoter interactions and provide insights into the molecular mechanisms by which communication between enhancers and promoters is regulated.

The first high-quality chromosome-level genome of the Sipuncula Sipunculus nudus using HiFi and Hi-C data

Sipuncula is a class of exocoelomic unsegmented animals whose evolutionary relationships are unresolved. The peanut worm Sipunculus nudus is a globally distributed, economically important species belonging to the class Sipuncula. Herein, we present the first high-quality chromosome-level assembly of S. nudus based on HiFi reads and high-resolution chromosome conformation capture (Hi-C) data. The assembled genome was 1,427 Mb, with a contig N50 length of 29.46 Mb and scaffold N50 length of 80.87 Mb. Approximately 97.91% of the genome sequence was anchored to 17 chromosomes. A BUSCO assessment showed that 97.7% of the expectedly conserved genes were present in the genome assembly. The genome was composed of 47.91% repetitive sequences, and 28,749 protein-coding genes were predicted. A phylogenetic tree demonstrated that Sipuncula belongs to Annelida and diverged from the common ancestor of Polychaeta. The high-quality chromosome-level genome of S. nudus will serve as a valuable reference for studies of the genetic diversity and evolution of Lophotrochozoa.

Chromosomal Microarray Study in Prader-Willi Syndrome.

A high-resolution chromosome microarray analysis was performed on 154 consecutive individuals enrolled in the DESTINY PWS clinical trial for Prader-Willi syndrome (PWS). Of these 154 PWS individuals, 87 (56.5%) showed the typical 15q11-q13 deletion subtypes, 62 (40.3%) showed non-deletion maternal disomy 15 and five individuals (3.2%) had separate unexpected microarray findings. For example, one PWS male had Klinefelter syndrome with segmental isodisomy identified in both chromosomes 15 and X. Thirty-five (40.2%) of 87 individuals showed typical larger 15q11-q13 Type I deletion and 52 individuals (59.8%) showed typical smaller Type II deletion. Twenty-four (38.7%) of 62 PWS individuals showed microarray patterns indicating either maternal heterodisomy 15 subclass or a rare non-deletion (epimutation) imprinting center defect. Segmental isodisomy 15 was seen in 34 PWS subjects (54.8%) with 15q26.3, 15q14 and 15q26.1 bands most commonly involved and total isodisomy 15 seen in four individuals (6.5%). In summary, we report on PWS participants consecutively enrolled internationally in a single clinical trial with high-resolution chromosome microarray analysis to determine and describe an unbiased estimate of the frequencies and types of genetic defects and address potential at-risk genetic disorders in those with maternal disomy 15 subclasses in the largest PWS cohort studied to date.

Reorganization of three-dimensional chromatin architecture in Medicago truncatula under phosphorus deficiency.

Emerging evidence reveals that the three-dimensional (3D) chromatin architecture plays a key regulatory role in various biological processes of plants. However, information on the 3D chromatin architecture of the legume model plant Medicago truncatula and its potential roles in the regulation of response to mineral nutrient deficiency are very limited. Using high-resolution chromosome conformation capture sequencing, we identified the 3D genome structure of M. truncatula in terms of A/B compartments, topologically associated domains (TADs) and chromatin loops. The gene density, expressional level, and active histone modification were higher in A compartments than in B compartments. Moreover, we analysed the 3D chromatin architecture reorganization in response to phosphorus (P) deficiency. The intra-chromosomal cis-interaction proportion was increased by P deficiency, and a total of 748 A/B compartment switch regions were detected. In these regions, density changes in H3K4me3 and H3K27ac modifications were associated with expression of P deficiency-responsive genes involved in root system architecture and hormonal responses. Furthermore, these genes enhanced P uptake and mobilization by increasing root surface area and strengthening signal transduction under P deficiency. These findings advance our understanding of the potential roles of 3D chromatin architecture in responses of plants in general, and in particular in M. truncatula, to P deficiency.

KEMR-Net: A Knowledge-Enhanced Mask Refinement Network for Chromosome Instance Segmentation

This article proposes a mask refinement method for chromosome instance segmentation. The proposed method exploits the knowledge representation capability of Neural Knowledge DNA (NK-DNA) to capture the semantics of the chromosome’s shape, texture, and key points, and then it uses the captured knowledge to improve the accuracy and smoothness of the masks. We validate the method’s effectiveness on our latest high-resolution chromosome image dataset. The experimental results show that our proposed method’s mask average precision (MaskAP) is 3.66% higher than Mask R-CNN and outperforms advanced Cascade Mask R-CNN by 1.35%.

Constitutional pericentric inversion of chromosome 16, inv(16)(p13.1q22), mimicking acute myeloid leukemia.

An acquired abnormality, inv(16)(p13q22), is a hallmark of acute myeloid leukemia with eosinophilia (AML M4Eo), which leads to leukemogenesis by forming the CBFB::MYH11 fusion gene [1]. We experienced a case of inv(16)(p13.1q22) discovered incidentally during bone marrow examination without evidence of AML. A 64-year-old woman was referred to our hospital because of hypergammaglobulinemia. She was being treated for rheumatoid arthritis, but had no past and family history of AML. Laboratory examination showed polyclonal gammopathy but no significant abnormalities in complete blood count with differential. Bone marrow picture showed a normocellular marrow and mild increase in plasma cells to 3.8% (reference range, 0.2%–1.7%), but no evidence of hematological malignancy. Surprisingly, chromosome analysis of the bone marrow cells revealed that all 20 cells had inv(16)(p13.1q22) mimicking AML M4Eo (Figure 1A). We therefore performed fluorescence in situ hybridization (FISH) analysis of the bone marrow cells by using the Vysis CBFB Dual Color Break Apart Rearrangement Probe (Abbott Molecular Diagnostics, Des Plaines, IL, USA). The splitting of the red and green signals reflecting truncation of the CBFB locus on chromosome 16q22 was not observed (Figure 1B). In addition, a multiplex reverse transcriptase-polymerase chain reaction assay covering 28 leukemia-specific fusion transcripts including CBFB::MYH11 was performed by using the HemaVision-28Q kit (VERITAS, Tokyo, Japan), but none of them was detected. Therefore, it was suggested that the chromosome inversion of this patient did not disrupt the CBFB gene. High-resolution chromosome analysis of the phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes also revealed inv(16)(p13.1q22) in all 20 cells, indicating a constitutional abnormality of chromosome 16 (Figure 1C). Thus, this patient was not misdiagnosed as AML M4Eo and has been followed up without treatment. To the best of our knowledge, only a few cases with inv(16)(p13q22) unrelated to AML have been reported [2-5]. All of these were concluded to be constitutional abnormalities, and some cases have been confirmed as familial onset. In these reports, there was no misdiagnosis of AML M4Eo in individuals without hematological malignancy, and in the case of chronic myeloid leukemia with t(9;22)(q34;q11) and inv(16)(p13q22), FISH analysis showed no abnormality of the MYH11 gene and inv(16)(p13q22) was also found in her healthy father, which avoided misdiagnosis as an additional cytogenetic abnormality in chronic phase [4]. Therefore, although extremely rare, we should be aware of the presence of constitutional chromosome abnormalities mimicking hematological malignancies, and it is important to perform further tests such as FISH and genomic analysis for differential diagnosis. We thank Yoshiko Sato, MT (LSI Medience Corporation, Tokyo, Japan), for her technical support. The authors declare that there is no conflict of interest. All procedures in this study were performed in accordance with the principles of the Declaration of Helsinki and the institutional guidelines. Informed consent was obtained from the patient and her family. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Genetic analysis of a child with Pitt-Hopkins syndrome due to a 18q21.2q21.32 deletion

To explore the genetic etiology of a child featuring global developmental and mental retardation. Chromosome G-banding karyotype analysis, copy number variation sequencing (CNV-seq) and high-resolution chromosome banding were used to screen the genomic variant in the child and his parents. Both the child and his father were found to have a karyotype of 46,XY,del(18)(q21.1q21.3), whilst his mother was 46,XX. CNV-seq analysis showed that the child was arr[19]18q21.2-q21.32(chr18:48 422 190-58 039 582)×1, with a 10.58 Mb deletion which encompassed the TCF4 gene. The same deletion was found in neither parent. High-resolution banding revealed that the father has a fragment of 18q21.1q21.3 inserted into 5p13.1. The child was diagnosed with Pitt-Hopkins syndrome due to the 18q21.2q21.32 deletion. Chromosome karyotyping and CNV-seq can effectively identify submicroscopic chromosome anomalies.

A review and performance evaluation of clustering frameworks for single-cell Hi-C data.

The three-dimensional genome structure plays a key role in cellular function and gene regulation. Single-cell Hi-C (high-resolution chromosome conformation capture) technology can capture genome structure information at the cell level, which provides the opportunity to study how genome structure varies among different cell types. Recently, a few methods are well designed for single-cell Hi-C clustering. In this manuscript, we perform an in-depth benchmark study of available single-cell Hi-C data clustering methods to implement an evaluation system for multiple clustering frameworks based on both human and mouse datasets. We compare eight methods in terms of visualization and clustering performance. Performance is evaluated using four benchmark metrics including adjusted rand index, normalized mutual information, homogeneity and Fowlkes-Mallows index. Furthermore, we also evaluate the eight methods for the task of separating cells at different stages of the cell cycle based on single-cell Hi-C data.

Single-cell Sequencing Reveals Clearance of Blastula Chromosomal Mosaicism in In Vitro Fertilization Babies

Although chromosomal mosaic embryos detected by trophectoderm (TE) biopsy offer healthy embryos available for transfer, high-resolution postnatal karyotyping and chromosome testing of the transferred embryos are insufficient. Here, we applied single-cell multi-omics sequencing for seven infants with blastula chromosomal mosaicism detected by TE biopsy. The chromosome ploidy was examined by single-cell genome analysis, with the cellular identity being identified by single-cell transcriptome analysis. A total of 1616 peripheral leukocytes from seven infants with embryonic chromosomal mosaicism and three control ones with euploid TE biopsy were analyzed. A small number of blood cells showed copy number alterations (CNAs) on seemingly random locations at a frequency of 0%−2.5% per infant. However, none of the cells showed CNAs that were the same as those of the corresponding TE biopsies. The blastula chromosomal mosaicism may be fully self-corrected, probably through the selective loss of the aneuploid cells during development, and the transferred embryos can be born as euploid infants without mosaic CNAs corresponding to the TE biopsies. The results provide a new reference for the evaluations of transferring chromosomal mosaic embryos in certain situations.

MCM complexes are barriers that restrict cohesin-mediated loop extrusion

Eukaryotic genomes are compacted into loops and topologically associating domains (TADs)1–3, which contribute to transcription, recombination and genomic stability4,5. Cohesin extrudes DNA into loops that are thought to lengthen until CTCF boundaries are encountered6–12. Little is known about whether loop extrusion is impeded by DNA-bound machines. Here we show that the minichromosome maintenance (MCM) complex is a barrier that restricts loop extrusion in G1 phase. Single-nucleus Hi-C (high-resolution chromosome conformation capture) of mouse zygotes reveals that MCM loading reduces CTCF-anchored loops and decreases TAD boundary insulation, which suggests that loop extrusion is impeded before reaching CTCF. This effect extends to HCT116 cells, in which MCMs affect the number of CTCF-anchored loops and gene expression. Simulations suggest that MCMs are abundant, randomly positioned and partially permeable barriers. Single-molecule imaging shows that MCMs are physical barriers that frequently constrain cohesin translocation in vitro. Notably, chimeric yeast MCMs that contain a cohesin-interaction motif from human MCM3 induce cohesin pausing, indicating that MCMs are ‘active’ barriers with binding sites. These findings raise the possibility that cohesin can arrive by loop extrusion at MCMs, which determine the genomic sites at which sister chromatid cohesion is established. On the basis of in vivo, in silico and in vitro data, we conclude that distinct loop extrusion barriers shape the three-dimensional genome.

SRAS-net: Low-resolution chromosome image classification based on deep learning.

Prenatal karyotype diagnosis is important to determine if the foetus has genetic diseases and some congenital diseases. Chromosome classification is an important part of karyotype analysis, and the task is tedious and lengthy. Chromosome classification methods based on deep learning have achieved good results, but if the quality of the chromosome image is not high, these methods cannot learn image features well, resulting in unsatisfactory classification results. Moreover, the existing methods generally have a poor effect on sex chromosome classification. Therefore, in this work, the authors propose to use a super‐resolution network, Self‐Attention Negative Feedback Network, and combine it with traditional neural networks to obtain an efficient chromosome classification method called SRAS‐net. The method first inputs the low‐resolution chromosome images into the super‐resolution network to generate high‐resolution chromosome images and then uses the traditional deep learning model to classify the chromosomes. To solve the problem of inaccurate sex chromosome classification, the authors also propose to use the SMOTE algorithm to generate a small number of sex chromosome samples to ensure a balanced number of samples while allowing the model to learn more sex chromosome features. Experimental results show that our method achieves 97.55% accuracy and is better than state‐of‐the‐art methods.

The genome organization of Neurospora crassa at high resolution uncovers principles of fungal chromosome topology.

The eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high-resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassa at a high resolution. Our composite Hi-C dataset, which merges 2 independent datasets generated with restriction enzymes that monitor euchromatin (DpnII) and heterochromatin (MseI), along with our DpnII/MseI double digest dataset, provide exquisite detail for both the conformation of entire chromosomes and the folding of chromatin at the resolution of individual genes. Within constitutive heterochromatin, we observe strong yet stochastic internal contacts, while euchromatin enriched with either activating or repressive histone post-translational modifications associates with constitutive heterochromatic regions, suggesting intercompartment contacts form to regulate transcription. Consistent with this, a strain with compromised heterochromatin experiences numerous changes in gene expression. Our high-resolution Neurospora Hi-C datasets are outstanding resources to the fungal community and provide valuable insights into higher organism genome topology.

Exploring the effects of genetic variation on gene regulation in cancer in the context of 3D genome structure

BackgroundNumerous genome-wide association studies (GWAS) conducted to date revealed genetic variants associated with various diseases, including breast and prostate cancers. Despite the availability of these large-scale data, relatively few variants have been functionally characterized, mainly because the majority of single-nucleotide polymorphisms (SNPs) map to the non-coding regions of the human genome. The functional characterization of these non-coding variants and the identification of their target genes remain challenging.ResultsIn this communication, we explore the potential functional mechanisms of non-coding SNPs by integrating GWAS with the high-resolution chromosome conformation capture (Hi-C) data for breast and prostate cancers. We show that more genetic variants map to regulatory elements through the 3D genome structure than the 1D linear genome lacking physical chromatin interactions. Importantly, the association of enhancers, transcription factors, and their target genes with breast and prostate cancers tends to be higher when these regulatory elements are mapped to high-risk SNPs through spatial interactions compared to simply using a linear proximity. Finally, we demonstrate that topologically associating domains (TADs) carrying high-risk SNPs also contain gene regulatory elements whose association with cancer is generally higher than those belonging to control TADs containing no high-risk variants.ConclusionsOur results suggest that many SNPs may contribute to the cancer development by affecting the expression of certain tumor-related genes through long-range chromatin interactions with gene regulatory elements. Integrating large-scale genetic datasets with the 3D genome structure offers an attractive and unique approach to systematically investigate the functional mechanisms of genetic variants in disease risk and progression.

A DAPI-Based Modified C-banding Technique for a Rapid Achieving High Photographic Contrast of Centromeres on Chromosomes.

Many chromosome assays rely on the quantification of chromosome abnormalities in cells, and one important abnormality is the existence of more than one centromere for each chromosome. The quantification of such abnormalities has been studied before. However, this process is labor-intensive and time consuming. Thus, this assay is challenging for ex-laboratory applications, where speed is required. We present a visualization method that uses a cheap stain-DAPI, long (e.g., high-resolution) chromosomes and our modified C-banding method for labeling chromosomes. The labeled chromosomes can then be easily seen with a conventional and readily available fluorescence microscopy system. This method achieves an acceleration of the detection of the presence of constitutive heterochromatin in chromosomal centromeres by more than 10 times, to ~2 h, in Human lymphocyte cells and in cells of the human Jurkat line. This new procedure will ultimately provide an easier and cheaper alternative to FISH/PNA probes, or the classic Giemsa staining method. Simplification and reduction in time of the overall procedure will enable the utilization of centromere-counting assays in laboratory and ex-laboratory applications, including in emergency response.

Genome organization controls transcriptional dynamics during development.

Past studies offer contradictory claims for the role of genome organization in the regulation of gene activity. Here, we show through high-resolution chromosome conformation analysis that the Drosophila genome is organized by two independent classes of regulatory sequences, tethering elements and insulators. Quantitative live imaging and targeted genome editing demonstrate that this two-tiered organization is critical for the precise temporal dynamics of Hox gene transcription during development. Tethering elements mediate long-range enhancer-promoter interactions and foster fast activation kinetics. Conversely, the boundaries of topologically associating domains (TADs) prevent spurious interactions with enhancers and silencers located in neighboring TADs. These two levels of genome organization operate independently of one another to ensure precision of transcriptional dynamics and the reliability of complex patterning processes.

Analysis of autosomal dominant genes impacted by copy number loss in 24,844 fetuses without structural abnormalities

BackgroundThe broad application of high-resolution chromosome detection technology in prenatal diagnosis has identified copy number loss (CNL) involving autosomal dominant (AD) genes in certain fetuses. Exon sequencing of fetuses exhibiting structural anomalies yields diagnostic information in up to 20% of cases. However, there is currently no relevant literature about the genetic origin and pregnancy outcome of CNL involving AD genes in fetuses without structural abnormalities.ResultsThis was a prospective study involving pregnant women who underwent amniocentesis for fetal copy number variation sequencing (CNVseq). Detection of parent-of-origin was suggested in cases of samples with CNL involving AD genes and the pregnancy outcome was monitored. Amniotic fluid samples from 24,844 fetuses without structural abnormalities were successfully tested via CNVseq. The results showed that 134 fetuses (0.5%) had small CNL (< 10 Mb) containing AD genes, after excluding microdeletion and microduplication syndrome and polymorphisms. By monitoring the pregnancy outcomes of the 134 fetuses, we found that 104 (77.6%) were good, 13 (9.7%) were adverse, and 17 (12.7%) pregnant women voluntarily chose to terminate pregnancy. Of the 13 fetuses with adverse pregnancy outcomes, only 2 fetuses had phenotypes consistent with those of diseases caused by AD genes involved in CNL.ConclusionsThe overall prognosis for fetuses without family history or structural abnormalities but with small CNL containing AD genes detected during pregnancy is good. The genetic origin, overlap status of established haploinsufficient gene and/or region, size of the CNL, and genetic mode may affect the pathogenicity of the CNL.

Capture-C: a modular and flexible approach for high-resolution chromosome conformation capture.

Chromosome conformation capture (3C) methods measure the spatial proximity between DNA elements in the cell nucleus. Many methods have been developed to sample 3C material, including the Capture-C family of protocols. Capture-C methods use oligonucleotides to enrich for interactions of interest from sequencing-ready 3C libraries. This approach is modular and has been adapted and optimised to work for sampling of disperse DNA elements (NuTi Capture-C), including from low-cell inputs (LI Capture-C), as well as to generate Hi-C like maps for specific regions of interest (Tiled-C) and to interrogate multi-way interactions (Tri-C). We present the design, experimental protocol and analysis pipeline for NuTi Capture-C in addition to the variations for generation of LI Capture-C, Tiled-C and Tri-C data. The entire procedure can be performed in three weeks and requires standard molecular biology skills and equipment, access to a next-generation sequencing platform, and basic bioinformatic skills. Implemented with other sequencing technologies, these methods can be used to identify regulatory interactions and to compare the structural organisation of the genome in different cell types and genetic models.