High-throughput Chromosome Conformation Capture Research Articles

The sequencing and de novo assembly of the Larimichthys crocea genome using PacBio and Hi-C technologies

Larimichthys crocea is an endemic marine fish in East Asia that belongs to Sciaenidae in Perciformes. L. crocea has now been recognized as an “iconic” marine fish species in China because not only is it a popular food fish in China, it is a representative victim of overfishing and still provides high value fish products supported by the modern large-scale mariculture industry. Here, we report a chromosome-level reference genome of L. crocea generated by employing the PacBio single molecule sequencing technique (SMRT) and high-throughput chromosome conformation capture (Hi-C) technologies. The genome sequences were assembled into 1,591 contigs with a total length of 723.86 Mb and a contig N50 length of 2.83 Mb. After chromosome-level scaffolding, 24 scaffolds were constructed with a total length of 668.67 Mb (92.48% of the total length). Genome annotation identified 23,657 protein-coding genes and 7262 ncRNAs. This highly accurate, chromosome-level reference genome of L. crocea provides an essential genome resource to support the development of genome-scale selective breeding and restocking strategies of L. crocea.

P2864A novel mechanism of sinus node dysfunction: intergenic deletion between PITX2 and ANK2 disrupts chromatin structure in pacemaker cell differentiation

Abstract Background Genetics of sinus node dysfunction (SND) remains poorly understood with few genes identified (HCN4, SCN5A, GJA5, KCNQ1, MYH6 and ANK2) and further genetic heterogeneity. Purpose We report two families with SND segregating with an intergenic deletion associated with long-range cis-regulatory elements (CREs) of PITX2. Methods We applied 30x whole genome sequencing (WGS) to two French families in which exome sequencing did not allow to detect the causing genes of SND and analyzed them bioinformatically. Results We first applied WGS to a 4-generation family presenting 16 patients with SND, atrial fibrillation and/or repolarization abnormalities and identified a 15-Kb deletion within a 16.5-cM linkage interval (Zmax = 5.9, θ = 0) on chromosome 4q25. We also applied WGS on a second family presenting 5 patients with SND and identified a 91-Kb deletion overlapping the initial deletion. This intergenic deletion, which was located between PITX2 and ANK2 and contained a binding motif of CCCTC-binding factor (CTCF), was segregating in all patients and was not found in 855 French control genomes. CTCF functions as a genome organizer mediating genomic interactions between genes and CREs. To clarify a possible regulatory function of the 1.5-Mb intergenic region containing the deletion, we interrogated 3 epigenetic databases. High-throughput chromosome conformation capture (Hi-C) (1) revealed that this intergenic region was placed in a topologically associating domain (TAD) containing PITX2. Chromatin interaction analysis by paired-end tag (ChIA-PET) of CTCF (2) suggested several possible functional chromatin loops in the TAD. Of them, a CTCF-mediated chromatin loop corresponding to a 300-Kb genomic region was clarified as a possible CRE using histone modification data from Roadmap. The region ranging between the intra-deletion CTCF motif and another distal motif was repressed in H3K27me3 profiles of fetal heart, while the region was activated in H3K27ac profiles of H1 ESC-derived mesendoderm. Conclusion This CRE may regulate expression of PITX2 and/or ANK2 in pacemaker cell differentiation. Ongoing experiments on patient's iPSC and transgenic mouse will further characterize the disease mechanism.

Chromosome-level assembly of the mustache toad genome using third-generation DNA sequencing and Hi-C analysis.

BackgroundThe mustache toad, Vibrissaphora ailaonica, is endemic to China and belongs to the Megophryidae family. Like other mustache toad species, V. ailaonica males temporarily develop keratinized nuptial spines on their upper jaw during each breeding season, which fall off at the end of the breeding season. This feature is likely result of the reversal of sexual dimorphism in body size, with males being larger than females. A high-quality reference genome for the mustache toad would be invaluable to investigate the genetic mechanism underlying these repeatedly developing keratinized spines.FindingsTo construct the mustache toad genome, we generated 225 Gb of short reads and 277 Gb of long reads using Illumina and Pacific Biosciences (PacBio) sequencing technologies, respectively. Sequencing data were assembled into a 3.53-Gb genome assembly, with a contig N50 length of 821 kb. We also used high-throughput chromosome conformation capture (Hi-C) technology to identify contacts between contigs, then assembled contigs into scaffolds and assembled a genome with 13 chromosomes and a scaffold N50 length of 412.42 Mb. Based on the 26,227 protein-coding genes annotated in the genome, we analyzed phylogenetic relationships between the mustache toad and other chordate species. The mustache toad has a relatively higher evolutionary rate and separated from a common ancestor of the marine toad, bullfrog, and Tibetan frog 206.1 million years ago. Furthermore, we identified 201 expanded gene families in the mustache toad, which were mainly enriched in immune pathway, keratin filament, and metabolic processes.ConclusionsUsing Illumina, PacBio, and Hi-C technologies, we constructed the first high-quality chromosome-level mustache toad genome. This work not only offers a valuable reference genome for functional studies of mustache toad traits but also provides important chromosomal information for wider genome comparisons.

Whole-genome sequencing and analysis of the Chinese herbal plant Gelsemium elegans

BackgroundGelsemium elegans (G. elegans) (2n = 2x = 16) is genus of flowering plants belonging to the Gelsemicaeae family. MethodHere, a high-quality genome assembly using the Oxford Nanopore Technologies (ONT) platform and high-throughput chromosome conformation capture techniques (Hi-C) were used. ResultsA total of 56.11 Gb of raw GridION X5 platform ONT reads (6.23 Gb per cell) were generated. After filtering, 53.45 Gb of clean reads were obtained, giving 160 × coverage depth. The de novo genome assemblies 335.13 Mb, close to the 338 Mb estimated by k-mer analysis, was generated with contig N50 of 10.23 Mb. The vast majority (99.2%) of the G. elegans assembled sequence was anchored onto 8 pseudo-chromosomes. The genome completeness was then evaluated and 1338 of the 1440 conserved genes (92.9%) could be found in the assembly. Genome annotation revealed that 43.16% of the G. elegans genome is composed of repetitive elements and 23.9% is composed of long terminal repeat elements. We predicted 26,768 protein-coding genes, of which 84.56% were functionally annotated. ConclusionThe genomic sequences of G. elegans could be a valuable source for comparative genomic analysis in the Gelsemicaeae family and will be useful for understanding the phylogenetic relationships of the indole alkaloid metabolism.

Assembly of allele-aware, chromosomal-scale autopolyploid genomes based on Hi-C data.

Construction of chromosome-level assembly is a vital step in achieving the goal of a 'Platinum' genome, but it remains a major challenge to assemble and anchor sequences to chromosomes in autopolyploid or highly heterozygous genomes. High-throughput chromosome conformation capture (Hi-C) technology serves as a robust tool to dramatically advance chromosome scaffolding; however, existing approaches are mostly designed for diploid genomes and often with the aim of reconstructing a haploid representation, thereby having limited power to reconstruct chromosomes for autopolyploid genomes. We developed a novel algorithm (ALLHiC) that is capable of building allele-aware, chromosomal-scale assembly for autopolyploid genomes using Hi-C paired-end reads with innovative 'prune' and 'optimize' steps. Application on simulated data showed that ALLHiC can phase allelic contigs and substantially improve ordering and orientation when compared to other mainstream Hi-C assemblers. We applied ALLHiC on an autotetraploid and an autooctoploid sugar-cane genome and successfully constructed the phased chromosomal-level assemblies, revealing allelic variations present in these two genomes. The ALLHiC pipeline enables de novo chromosome-level assembly of autopolyploid genomes, separating each allele. Haplotype chromosome-level assembly of allopolyploid and heterozygous diploid genomes can be achieved using ALLHiC, overcoming obstacles in assembling complex genomes.

A high-quality genome assembly for the endangered golden snub-nosed monkey (Rhinopithecus roxellana).

BackgroundThe golden snub-nosed monkey (Rhinopithecus roxellana) is an endangered colobine species endemic to China, which has several distinct traits including a unique social structure. Although a genome assembly for R. roxellana is available, it is incomplete and fragmented because it was constructed using short-read sequencing technology. Thus, important information such as genome structural variation and repeat sequences may be absent.FindingsTo obtain a high-quality chromosomal assembly for R. roxellana qinlingensis, we used 5 methods: Pacific Bioscience single-molecule real-time sequencing, Illumina paired-end sequencing, BioNano optical maps, 10X Genomics link-reads, and high-throughput chromosome conformation capture. The assembled genome was ∼3.04 Gb, with a contig N50 of 5.72 Mb and a scaffold N50 of 144.56 Mb. This represented a 100-fold improvement over the previously published genome. In the new genome, 22,497 protein-coding genes were predicted, of which 22,053 were functionally annotated. Gene family analysis showed that 993 and 2,745 gene families were expanded and contracted, respectively. The reconstructed phylogeny recovered a close relationship between R. rollexana and Macaca mulatta, and these 2 species diverged ∼13.4 million years ago.ConclusionWe constructed a high-quality genome assembly of the Qinling golden snub-nosed monkey; it had superior continuity and accuracy, which might be useful for future genetic studies in this species and as a new standard reference genome for colobine primates. In addition, the updated genome assembly might improve our understanding of this species and could assist conservation efforts.

Non-coding variability at the APOE locus contributes to the Alzheimer\u2019s risk

Alzheimer’s disease (AD) is a leading cause of mortality in the elderly. While the coding change of APOE-ε4 is a key risk factor for late-onset AD and has been believed to be the only risk factor in the APOE locus, it does not fully explain the risk effect conferred by the locus. Here, we report the identification of AD causal variants in PVRL2 and APOC1 regions in proximity to APOE and define common risk haplotypes independent of APOE-ε4 coding change. These risk haplotypes are associated with changes of AD-related endophenotypes including cognitive performance, and altered expression of APOE and its nearby genes in the human brain and blood. High-throughput genome-wide chromosome conformation capture analysis further supports the roles of these risk haplotypes in modulating chromatin states and gene expression in the brain. Our findings provide compelling evidence for additional risk factors in the APOE locus that contribute to AD pathogenesis.

Abstract 5208: Genome-wide AR enhancer activity in prostate cancer

Abstract Prostate cancer (PCa) is one of the most commonly diagnosed forms of cancer with one out of every nine North American men developing the disease in their lifetime. Despite robust early detection methods, new therapeutics, and advancements in surgical operations, PCa is still second leading cause of cancer death in men. An increasing number of studies have shown that non-coding mutations plays a critical role in the development and progression of PCa. These somatic mutations can disrupt regulatory elements such as enhancers and alter the transcriptional landscape of the cancer. Discovering and interpreting the non-coding elements is crucial to understand the nature of this disease. At all stages of PCa, androgen receptor (AR) signaling is essential to the cancer. Recent work from our laboratory demonstrated that there is a significant increase in somatic mutations at AR binding sites (ARBS). Given the importance of AR-mediated transcription, as well as the recurrent nature of these mutations, we proposed that these somatic mutations could play an important role in PCa development. To stratify the mutations, we conducted a massively multi-parallel enhancer assay to identify those ARBS sites with enhancer activity. With STARR-seq we found that 8% of the ARBS had strong inducible AR enhancer activity (341/4139). Interestingly, the majority of these sites did not contain a canonical androgen response element. With these results we incorporated high-throughput chromosome conformation capture (HiC) to formally define the genes regulated by each of these AR enhancers. Based on these results we have selected and characterized several non-coding ARBS mutations. We believe that the importance of the regulatory role of these regions will play a tremendous role in understanding of the prostate cancer and its progression. Citation Format: Dogancan Ozturan, Flora Huang, Tunc Morova, Mohammadali Saffarzadeh, Nathan A. Lack. Genome-wide AR enhancer activity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5208.

Abstract 1654: Detection of structural variations in a cancer reference sample with multiple NGS platforms

Abstract Structural Variations (SVs) are genetic variations or rearrangements in the structure of chromosome. SVs study plays an important role in cancer research as cancer genome is commonly altered with thousands of structural rearrangements such as insertions, deletions, translocation, inversions, duplications, copy number variations (CNVs). Next-generation sequencing (NGS) technology has provided a cost-effective approach for the comprehensive detection of SVs in human cancer cells. In this study, we systematically investigated the somatic structural variations in a pair of breast cancer cell lines with multiple NGS platforms including Illumina short-read, 10x genomics linked-read, PacBio long-read, and high-throughput chromosome conformation capture HiC. We systematically evaluated the factors that impact the SVs detection accuracy. We measured the reproductivity of somatic SV detections across platforms and benchmarked the different software tools’ performance. We performed SV integration from different platforms and established a high-confident SV call set for the reference samples. To independently evaluate the accuracy of our SV call set, we used orthogonal methods such as cytogenetic array as well as fusion gene detected from RNA-seq for cross validation. From this study, we identified the unique strengths of each NGS technologies and provide a practical guide to integrate multiple platform data to improve SV detection sensitivity and accuracy in cancer genome. Note: This abstract was not presented at the meeting. Citation Format: Yongmei Zhao, Wenming Xiao, on behalf of Somatic Mutation Working Group of theSEQC2 Consortium. Detection of structural variations in a cancer reference sample with multiple NGS platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1654.

The effects of Arabidopsis genome duplication on the chromatin organization and transcriptional regulation.

Autopolyploidy is widespread in higher plants and important for agricultural yield and quality. However, the effects of genome duplication on the chromatin organization and transcriptional regulation are largely unknown in plants. Using High-throughput Chromosome Conformation Capture (Hi-C), we showed that autotetraploid Arabidopsis presented more inter-chromosomal interactions and fewer short-range chromatin interactions compared with its diploid progenitor. In addition, genome duplication contributed to the switching of some loose and compact structure domains with altered H3K4me3 and H3K27me3 histone modification status. 539 genes were identified with altered transcriptions and chromatin interactions in autotetraploid Arabidopsis. Especially, we found that genome duplication changed chromatin looping and H3K27me3 histone modification in Flowering Locus C. We propose that genome doubling modulates the transcription genome-wide by changed chromatin interactions and at the specific locus by altered chromatin loops and histone modifications.

A chromosome-scale genome assembly of cucumber (Cucumis sativus L.).

BackgroundAccurate and complete reference genome assemblies are fundamental for biological research. Cucumber is an important vegetable crop and model system for sex determination and vascular biology. Low-coverage Sanger sequences and high-coverage short Illumina sequences have been used to assemble draft cucumber genomes, but the incompleteness and low quality of these genomes limit their use in comparative genomics and genetic research. A high-quality and complete cucumber genome assembly is therefore essential.FindingsWe assembled single-molecule real-time (SMRT) long reads to generate an improved cucumber reference genome. This version contains 174 contigs with a total length of 226.2 Mb and an N50 of 8.9 Mb, and provides 29.0 Mb more sequence data than previous versions. Using 10X Genomics and high-throughput chromosome conformation capture (Hi-C) data, 89 contigs (∼211.0 Mb) were directly linked into 7 pseudo-chromosome sequences. The newly assembled regions show much higher guanine-cytosine or adenine-thymine content than found previously, which is likely to have been inaccessible to Illumina sequencing. The new assembly contains 1,374 full-length long terminal retrotransposons and 1,078 novel genes including 239 tandemly duplicated genes. For example, we found 4 tandemly duplicated tyrosylprotein sulfotransferases, in contrast to the single copy of the gene found previously and in most other plants.ConclusionThis high-quality genome presents novel features of the cucumber genome and will serve as a valuable resource for genetic research in cucumber and plant comparative genomics.

Cis- and Trans-chromosomal Interactions Define Pericentric Boundaries in the Absence of Conventional Heterochromatin.

The diploid budding yeast Candida albicans harbors unique CENPA-rich 3- to 5-kb regions that form the centromere (CEN) core on each of its eight chromosomes. The epigenetic nature of these CENs does not permit the stabilization of a functional kinetochore on an exogenously introduced CEN plasmid. The flexible nature of such centromeric chromatin is exemplified by the reversible silencing of a transgene upon its integration into the CENPA-bound region. The lack of a conventional heterochromatin machinery and the absence of defined boundaries of CENPA chromatin makes the process of CEN specification in this organism elusive. Additionally, upon native CEN deletion, C. albicans can efficiently activate neocentromeres proximal to the native CEN locus, hinting at the importance of CEN-proximal regions. In this study, we examine this CEN-proximity effect and identify factors for CEN specification in C. albicans We exploit a counterselection assay to isolate cells that can silence a transgene when integrated into the CEN-flanking regions. We show that the frequency of reversible silencing of the transgene decreases from the central core of CEN7 to its peripheral regions. Using publicly available C. albicans high-throughput chromosome conformation capture data, we identify a 25-kb region centering on the CENPA-bound core that acts as CEN-flanking compact chromatin (CFCC). Cis- and trans-chromosomal interactions associated with the CFCC spatially segregates it from bulk chromatin. We further show that neocentromere activation on chromosome 7 occurs within this specified region. Hence, this study identifies a specialized CEN-proximal domain that specifies and restricts the centromeric activity to a unique region.

Advances in mammalian three-dimensional genome by using Hi-C technology approach

Mammalian genomic DNA in the cell nucleus doesn't exist in linear form but is highly folded and condensed into chromatin with a three-dimensional (3D) structure possessing a specific spatial structure and conformation. Hi-C, the high-throughput chromosome conformation capture technology, was first published in 2009, and it provides an in-depth view of 3D genomics. According to the size of DNA unit, the 3D hierarchical units of mammalian genome can be categorized sequentially as chromosome territory (CT), chromatin compartment A/B, topological associated domain (TAD), and chromatin loop. These hierarchical structural units play vital roles in gene transcription and regulation. In this review, we summarize the 3D hierarchical division of chromosomes, the effects of hierarchical units and the applications of Hi-C technology in development and disease. This review is intended to provide insights for the further study of 3D genomics in mammals.

Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations.

Balanced chromosomal rearrangements such as inversions and translocations can cause congenital disease or cancer by inappropriately rewiring promoter-enhancer contacts1,2. To study the potentially pathogenic consequences of balanced chromosomal rearrangements, we generated a series of genomic inversions by placing an active limb enhancer cluster from the Epha4 regulatory domain at different positions within a neighbouring gene-dense region and investigated their effects on gene regulation in vivo in mice. Expression studies and high-throughput chromosome conformation capture from embryonic limb buds showed that the enhancer cluster activated several genes downstream that are located within asymmetric regions of contact, the so-called architectural stripes3. The ectopic activation of genes led to a limb phenotype that could be rescued by deleting the CCCTC-binding factor (CTCF) anchor of the stripe. Architectural stripes appear to be driven by enhancer activity, because they do not form in mouse embryonic stem cells. Furthermore, we show that architectural stripes are a frequent feature of developmental three-dimensional genome architecture often associated with active enhancers. Therefore, balanced chromosomal rearrangements can induce ectopic gene expression and the formation of asymmetric chromatin contact patterns that are dependent on CTCF anchors and enhancer activity.

Abstract B099: Elucidating the mechanisms that underpin RAG chromatin scanning in V(D)J recombination at antigen receptor gene loci

Abstract Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons are assembled from V, D and J segments by V(D)J recombination, which is initiated by RAG1/RAG2 endonuclease (RAG) that introduces DNA double-stranded breaks (DSBs) between a pair of V, D, and J coding segments and flanking recombination signal sequences (RSSs). IgH V(D)J recombination is ordered, with D-JH joining occurring before appendage of a VH to a DJH intermediate in pro-B cells. In pre-B cells, a single-step VL-to-JL recombination event is invoked at IgL loci, with Ig kappa rearrangement usually preceding that of Ig lambda. The mechanisms of Ig V(D)J recombination, especially long-range VH-to-DJH and Vkappa-to-Jkappa recombination are still not fully understood. Recently, our work showed CTCF-binding elements (CBEs) immediately downstream of frequently rearranged VH-RSSs increase recombination potential of their associated VH far beyond that provided by RSS alone. This CBE activity becomes particularly striking upon the inactivation of IgH IGCR1 control element, which promotes DJH intermediate formation by insulting Ds, JHs, and recombination center (RC) from upstream VHs in a CBE-anchored chromatin loop. By developing and applying 3C-HTGTS, a new high-throughput chromosome conformation capture method that shows great advantages over classical 4C-seq in sensitivity and specificity, we found VH-associated CBEs promotes interaction with the RC. Based on these, we proposed a RAG chromatin scanning model for V(D)J recombination, in which VH CBEs stabilize interactions of D-proximal VHs first encountered by the DJH RC during linear RAG scanning on chromatin and thereby promote dominant rearrangement of these VHs by an unanticipated chromatin accessibility-enhancing CBE function. However, it is still not known if such chromatin scanning also happens on other antigen receptor loci and what the underlying mechanism is. Here, we will show our new findings on the roles of RAG chromatin scanning in Vkappa-to-Jkappa recombination at Ig kappa locus by multiple comprehensive approaches. Moreover, we will also discuss our most updated understanding of the mechanisms underlying RAG chromatin scanning using a novel and sophisticated approach. The potent mutagenic and oncogenic effects of aberrant RAG chromatin scanning at RAG “off-target” sites genome-wide will be also discussed. Citation Format: Zhaoqing Ba, Suvi Jain, Jiazhi Hu, Frederick Alt. Elucidating the mechanisms that underpin RAG chromatin scanning in V(D)J recombination at antigen receptor gene loci [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B099.

Chromosomal level assembly and population sequencing of the Chinese tree shrew genome.

Chinese tree shrews (Tupaia belangeri chinensis) have become an increasingly important experimental animal in biomedical research due to their close relationship to primates. An accurately sequenced and assembled genome is essential for understanding the genetic features and biology of this animal. In this study, we used long-read single-molecule sequencing and high-throughput chromosome conformation capture (Hi-C) technology to obtain a high-qualitychromosome-scale scaffolding of the Chinese tree shrew genome. The new reference genome (KIZ version 2: TS_2.0) resolved problems in presently available tree shrew genomes and enabled accurate identification of large and complex repeat regions, gene structures, and species-specific genomic structural variants. In addition, by sequencing the genomes of six Chinese tree shrew individuals, we produced a comprehensive map of 12.8 M single nucleotide polymorphisms and confirmed that the major histocompatibility complex (MHC) loci and immunoglobulin gene family exhibited high nucleotide diversity in the tree shrew genome. We updated the tree shrew genome database (TreeshrewDB v2.0: http://www.treeshrewdb.org) to include the genome annotation information and genetic variations. The new high-quality reference genome of the Chinese tree shrew and the updated TreeshrewDB will facilitate the use of this animal in many different fields of research.

Sub-nucleosomal Genome Structure Reveals Distinct Nucleosome Folding Motifs

Elucidating the global and local rules that govern genome-wide, hierarchical chromatin architecture remains a critical challenge. Current high-throughput chromosome conformation capture (Hi-C) technologies have identified large-scale chromatin structural motifs, such as topologically associating domains and looping. However, structural rules at the smallest or nucleosome scale remain poorly understood. Here, we coupled nucleosome-resolved Hi-C technology with simulated annealing-molecular dynamics (SA-MD) simulation to reveal 3D spatial distributions of nucleosomes and their genome-wide orientation in chromatin. Our method, called Hi-CO, revealed distinct nucleosome folding motifs across the yeast genome. Our results uncovered two types of basic secondary structural motifs in nucleosome folding: α-tetrahedron and β-rhombus analogous to α helix and β sheet motifs in protein folding. Using mutants and cell-cycle-synchronized cells, we further uncovered motifs with specific nucleosome positioning and orientation coupled to epigenetic features at individual loci. By illuminating molecular-level structure-function relationships in eukaryotic chromatin, our findings establish organizational principles of nucleosome folding.

Structural Variation Detection by Proximity Ligation from Formalin-Fixed, Paraffin-Embedded Tumor Tissue

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.

Reference genome sequences of two cultivated allotetraploid cottons, Gossypium hirsutum and Gossypium barbadense

Allotetraploid cotton species (Gossypium hirsutum and Gossypium barbadense) have long been cultivated worldwide for natural renewable textile fibers. The draft genome sequences of both species are available but they are highly fragmented and incomplete1–4. Here we report reference-grade genome assemblies and annotations for G. hirsutum accession Texas Marker-1 (TM-1) and G. barbadense accession 3–79 by integrating single-molecule real-time sequencing, BioNano optical mapping and high-throughput chromosome conformation capture techniques. Compared with previous assembled draft genomes1,3, these genome sequences show considerable improvements in contiguity and completeness for regions with high content of repeats such as centromeres. Comparative genomics analyses identify extensive structural variations that probably occurred after polyploidization, highlighted by large paracentric/pericentric inversions in 14 chromosomes. We constructed an introgression line population to introduce favorable chromosome segments from G. barbadense to G. hirsutum, allowing us to identify 13 quantitative trait loci associated with superior fiber quality. These resources will accelerate evolutionary and functional genomic studies in cotton and inform future breeding programs for fiber improvement.

Multiple transcription factors contribute to inter-chromosomal interaction in yeast

BackgroundChromatin interactions medicated by genomic elements located throughout the genome play important roles in gene regulation and can be identified with the technologies such as high-throughput chromosome conformation capture (Hi-C), followed by next-generation sequencing. These techniques were wildly used to reveal the relative spatial disposition of chromatins in human, mouse and yeast. Unlike metazoan where CTCF plays major roles in mediating chromatin interactions, in yeast, the transcription factors (TFs) involved in this biological process are poorly known.ResultsHere, we presented two computational approaches to estimate the TFs enriched in the chromatin physical inter-chromosomal interactions in yeast. Through the Chi-square method, we found TFs whose binding data are differentially distributed in different interaction groups, including Cin5, Stp1 and Sut1, whose binding data are negatively correlated with the chromosome spatial distance. A multivariate linear regression model was employed to estimate the potential contribution of different transcription factors against the physical distance of chromosomes. Rlr1, Set12 and Dig1 were found to be top positively participated in these chromosomal interactions. Ste12 was highlighted to be involved in gene reposition. Overall, we found 10 TFs enriched from both computational approaches, potentially to be involved in inter-chromosomal interactions.ConclusionsNo transcription factor (TF) in our study was found to have a dominant impact on the inter-chromosomal interaction as CTCF did in human or other metazoan, suggesting species without CTCF might have different regulatory systems in mediating inter-chromosomal interactions. In summary, we presented a systematic examination of TFs involved in chromatin interaction in yeast and the results provide candidate TFs for future studies.