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1.
Nat Commun ; 11(1): 5040, 2020 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-33028839

RESUMO

Bringing together cancer genomes from different projects increases power and allows the investigation of pan-cancer, molecular mechanisms. However, working with whole genomes sequenced over several years in different sequencing centres requires a framework to compare the quality of these sequences. We used the Pan-Cancer Analysis of Whole Genomes cohort as a test case to construct such a framework. This cohort contains whole cancer genomes of 2832 donors from 18 sequencing centres. We developed a non-redundant set of five quality control (QC) measurements to establish a star rating system. These QC measures reflect known differences in sequencing protocol and provide a guide to downstream analyses and allow for exclusion of samples of poor quality. We have found that this is an effective framework of quality measures. The implementation of the framework is available at: https://dockstore.org/containers/quay.io/jwerner_dkfz/pancanqc:1.2.2 .


Assuntos
Genoma Humano/genética , Genômica/normas , Neoplasias/genética , Controle de Qualidade , Mapeamento Cromossômico/normas , Cromossomos Humanos/genética , Análise Mutacional de DNA/normas , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Masculino , Mutação , Software , Sequenciamento Completo do Genoma/normas
2.
Hum Genomics ; 14(1): 36, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33036646

RESUMO

INTRODUCTION: The course of COVID-19 varies from asymptomatic to severe in patients. The basis for this range in symptoms is unknown. One possibility is that genetic variation is partly responsible for the highly variable response. We evaluated how well a genetic risk score based on chromosomal-scale length variation and machine learning classification algorithms could predict severity of response to SARS-CoV-2 infection. METHODS: We compared 981 patients from the UK Biobank dataset who had a severe reaction to SARS-CoV-2 infection before 27 April 2020 to a similar number of age-matched patients drawn for the general UK Biobank population. For each patient, we built a profile of 88 numbers characterizing the chromosomal-scale length variability of their germ line DNA. Each number represented one quarter of the 22 autosomes. We used the machine learning algorithm XGBoost to build a classifier that could predict whether a person would have a severe reaction to COVID-19 based only on their 88-number classification. RESULTS: We found that the XGBoost classifier could differentiate between the two classes at a significant level (p = 2 · 10-11) as measured against a randomized control and (p = 3 · 10-14) as measured against the expected value of a random guessing algorithm (AUC = 0.5). However, we found that the AUC of the classifier was only 0.51, too low for a clinically useful test. CONCLUSION: Genetics play a role in the severity of COVID-19, but we cannot yet develop a useful genetic test to predict severity.


Assuntos
Algoritmos , Betacoronavirus/isolamento & purificação , Aberrações Cromossômicas , Cromossomos Humanos/genética , Infecções por Coronavirus/diagnóstico , Aprendizado de Máquina , Pneumonia Viral/diagnóstico , Índice de Gravidade de Doença , Betacoronavirus/genética , Estudos de Casos e Controles , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/genética , Infecções por Coronavirus/virologia , Conjuntos de Dados como Assunto , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/genética , Pneumonia Viral/virologia , Fatores de Risco
3.
Nat Biotechnol ; 38(9): 1044-1053, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32686750

RESUMO

De novo assembly of a human genome using nanopore long-read sequences has been reported, but it used more than 150,000 CPU hours and weeks of wall-clock time. To enable rapid human genome assembly, we present Shasta, a de novo long-read assembler, and polishing algorithms named MarginPolish and HELEN. Using a single PromethION nanopore sequencer and our toolkit, we assembled 11 highly contiguous human genomes de novo in 9 d. We achieved roughly 63× coverage, 42-kb read N50 values and 6.5× coverage in reads >100 kb using three flow cells per sample. Shasta produced a complete haploid human genome assembly in under 6 h on a single commercial compute node. MarginPolish and HELEN polished haploid assemblies to more than 99.9% identity (Phred quality score QV = 30) with nanopore reads alone. Addition of proximity-ligation sequencing enabled near chromosome-level scaffolds for all 11 genomes. We compare our assembly performance to existing methods for diploid, haploid and trio-binned human samples and report superior accuracy and speed.


Assuntos
Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento por Nanoporos , Análise de Sequência de DNA/métodos , Algoritmos , Benchmarking , Cromossomos Humanos/genética , Aprendizado Profundo , Genômica , Antígenos HLA/genética , Haploidia , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Análise de Sequência de DNA/normas
4.
BMC Bioinformatics ; 21(Suppl 12): 304, 2020 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-32703240

RESUMO

BACKGROUND: The imputation of genotypes increases the power of genome-wide association studies. However, the imputation quality should be assessed in each particular case. Nevertheless, not all imputation softwares control the error of output, e.g., the last release of fastPHASE program (1.4.8) lacks such an option. In this particular software there is also an uncertainty in choosing the model parameters. fastPHASE is based on haplotype clusters, which size should be set a priori. The parameter influences the results of imputation and downstream analysis. RESULTS: We present a software toolkit imputeqc to assess the imputation quality and/or to choose the model parameters for imputation. We demonstrate the efficacy of toolkit for evaluation of imputations made with both fastPHASE and BEAGLE software for HapMap and 1000 Genomes data. The discordance of genotypes received correlated well in both methods. Using imputeqc, we also shown how to choose the optimal number of haplotype clusters and expectation-maximization cycles for fastPHASE program. The found number of haplotype clusters of 25 was further applied for hapFLK testing that revealed signatures of selection at LCT region on chromosome 2. We also demonstrated how to decrease the computational time in the case of hapFLK testing from 3 days to 20 h. CONCLUSIONS: The toolkit is implemented as an R package imputeqc and command line scripts. The code is freely available at https://github.com/inzilico/imputeqc under the MIT license.


Assuntos
Estudo de Associação Genômica Ampla , Software , Cromossomos Humanos/genética , Bases de Dados Genéticas , Genoma Humano , Genótipo , Haplótipos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Tamanho da Amostra
6.
Hum Genet ; 139(12): 1555-1563, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32535809

RESUMO

The chromosomal region critical in Down syndrome has long been analyzed through genotype-phenotype correlation studies using data from many patients with partial trisomy 21. Owing to that, a relatively small region of human chromosome 21 (35.9 ~ 38.0 Mb) has been considered as Down syndrome critical region (DSCR). In this study, microarray-based comparative genomic hybridization analysis identified complex rearrangements of chromosome 21 in a patient manifesting clinical features partially overlapped with that of Down syndrome. Although the patient did not show up-slanting palpebral fissures and single transverse palmar creases, other symptoms were consistent with Down syndrome. Rearrangements were analyzed by whole-genome sequencing using Nanopore long-read sequencing. The analysis revealed that chromosome 21 was fragmented into seven segments and reassembled by six connected points. Among 12 breakpoints, 5 are located within the short region and overlapped with repeated segments. The rearrangement resulted in a maximum gain of five copies, but no region showed loss of genomic copy numbers. Breakpoint-junctions showed no homologous region. Based on these findings, chromoanasynthesis was considered as the mechanism. Although the distal 21q22.13 region was not included in the aberrant regions, some of the genes located on the duplicated regions, SOD1, SON, ITSN1, RCAN1, and RUNX1, were considered as possible candidate genes for clinical features of the patient. We discussed the critical region for Down syndrome, with the literature review.


Assuntos
Cromossomos Humanos Par 21/genética , Cromossomos Humanos/genética , Síndrome de Down/genética , Proteínas Adaptadoras de Transporte Vesicular/genética , Aberrações Cromossômicas , Hibridização Genômica Comparativa/métodos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Proteínas de Ligação a DNA/genética , Síndrome de Down/fisiopatologia , Feminino , Dosagem de Genes/genética , Estudos de Associação Genética , Humanos , Hibridização in Situ Fluorescente/métodos , Lactente , Análise em Microsséries/métodos , Antígenos de Histocompatibilidade Menor/genética , Proteínas Musculares/genética , Superóxido Dismutase-1/genética , Sequenciamento Completo do Genoma
7.
Nature ; 584(7819): 130-135, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32581364

RESUMO

The extent to which the biology of oncogenesis and ageing are shaped by factors that distinguish human populations is unknown. Haematopoietic clones with acquired mutations become common with advancing age and can lead to blood cancers1-10. Here we describe shared and population-specific patterns of genomic mutations and clonal selection in haematopoietic cells on the basis of 33,250 autosomal mosaic chromosomal alterations that we detected in 179,417 Japanese participants in the BioBank Japan cohort and compared with analogous data from the UK Biobank. In this long-lived Japanese population, mosaic chromosomal alterations were detected in more than 35.0% (s.e.m., 1.4%) of individuals older than 90 years, which suggests that such clones trend towards inevitability with advancing age. Japanese and European individuals exhibited key differences in the genomic locations of mutations in their respective haematopoietic clones; these differences predicted the relative rates of chronic lymphocytic leukaemia (which is more common among European individuals) and T cell leukaemia (which is more common among Japanese individuals) in these populations. Three different mutational precursors of chronic lymphocytic leukaemia (including trisomy 12, loss of chromosomes 13q and 13q, and copy-neutral loss of heterozygosity) were between two and six times less common among Japanese individuals, which suggests that the Japanese and European populations differ in selective pressures on clones long before the development of clinically apparent chronic lymphocytic leukaemia. Japanese and British populations also exhibited very different rates of clones that arose from B and T cell lineages, which predicted the relative rates of B and T cell cancers in these populations. We identified six previously undescribed loci at which inherited variants predispose to mosaic chromosomal alterations that duplicate or remove the inherited risk alleles, including large-effect rare variants at NBN, MRE11 and CTU2 (odds ratio, 28-91). We suggest that selective pressures on clones are modulated by factors that are specific to human populations. Further genomic characterization of clonal selection and cancer in populations from around the world is therefore warranted.


Assuntos
Envelhecimento/genética , Aberrações Cromossômicas , Cromossomos Humanos/genética , Células Clonais/metabolismo , Genoma Humano/genética , Células-Tronco Hematopoéticas/metabolismo , Mutação , Idoso de 80 Anos ou mais , Alelos , Linhagem da Célula , Células Clonais/citologia , Células Clonais/patologia , Estudos de Coortes , Feminino , Loci Gênicos/genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/patologia , Humanos , Japão , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/patologia , Leucemia de Células T/genética , Leucemia de Células T/patologia , Masculino , Mosaicismo , Reino Unido
8.
Nature ; 582(7812): 432-437, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32499643

RESUMO

Highly structured RNA molecules usually interact with each other, and associate with various RNA-binding proteins, to regulate critical biological processes. However, RNA structures and interactions in intact cells remain largely unknown. Here, by coupling proximity ligation mediated by RNA-binding proteins with deep sequencing, we report an RNA in situ conformation sequencing (RIC-seq) technology for the global profiling of intra- and intermolecular RNA-RNA interactions. This technique not only recapitulates known RNA secondary structures and tertiary interactions, but also facilitates the generation of three-dimensional (3D) interaction maps of RNA in human cells. Using these maps, we identify noncoding RNA targets globally, and discern RNA topological domains and trans-interacting hubs. We reveal that the functional connectivity of enhancers and promoters can be assigned using their pairwise-interacting RNAs. Furthermore, we show that CCAT1-5L-a super-enhancer hub RNA-interacts with the RNA-binding protein hnRNPK, as well as RNA derived from the MYC promoter and enhancer, to boost MYC transcription by modulating chromatin looping. Our study demonstrates the power and applicability of RIC-seq in discovering the 3D structures, interactions and regulatory roles of RNA.


Assuntos
Conformação de Ácido Nucleico , RNA/química , RNA/genética , Análise de Sequência de RNA/métodos , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Cromossomos Humanos/genética , Elementos Facilitadores Genéticos/genética , Genes myc/genética , Genes de RNAr/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Reprodutibilidade dos Testes , Transcrição Genética
9.
Proc Natl Acad Sci U S A ; 117(22): 12131-12142, 2020 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-32414923

RESUMO

Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.


Assuntos
Estruturas Cromossômicas/química , Cromossomos Humanos/química , DNA Topoisomerases Tipo II/metabolismo , Heterocromatina/química , Mitose , Segregação de Cromossomos , Estruturas Cromossômicas/genética , Cromossomos Humanos/genética , Citocinese , DNA Topoisomerases Tipo II/genética , Células HCT116 , Heterocromatina/genética , Humanos , Metáfase
10.
Mol Cell ; 78(4): 714-724.e5, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32353258

RESUMO

Nonrandom DNA segregation (NDS) is a mitotic event in which sister chromatids carrying the oldest DNA strands are inherited exclusively by one of the two daughter cells. Although this phenomenon has been observed across various organisms, the mechanism and physiological relevance of this event remain poorly defined. Here, we demonstrate that DNA replication stress can trigger NDS in human cells. This biased inheritance of old template DNA is associated with the asymmetric DNA damage response (DDR), which derives at least in part from telomeric DNA. Mechanistically, we reveal that the ATR/CHK1 signaling pathway plays an essential role in mediating NDS. We show that this biased segregation process leads to cell-cycle arrest and cell death in damaged daughter cells inheriting newly replicated DNA. These data therefore identify a key role for NDS in the maintenance of genomic integrity within cancer cell populations undergoing replication stress due to oncogene activation.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Cromossomos Humanos/genética , Dano ao DNA , Replicação do DNA , Mitose , Proteínas Mutadas de Ataxia Telangiectasia/genética , Quinase 1 do Ponto de Checagem/genética , Segregação de Cromossomos , Células HeLa , Humanos , Transdução de Sinais
11.
Am J Hum Genet ; 106(6): 872-884, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32470376

RESUMO

Genome-wide analysis methods, such as array comparative genomic hybridization (CGH) and whole-genome sequencing (WGS), have greatly advanced the identification of structural variants (SVs) in the human genome. However, even with standard high-throughput sequencing techniques, complex rearrangements with multiple breakpoints are often difficult to resolve, and predicting their effects on gene expression and phenotype remains a challenge. Here, we address these problems by using high-throughput chromosome conformation capture (Hi-C) generated from cultured cells of nine individuals with developmental disorders (DDs). Three individuals had previously been identified as harboring duplications at the SOX9 locus and six had been identified with translocations. Hi-C resolved the positions of the duplications and was instructive in interpreting their distinct pathogenic effects, including the formation of new topologically associating domains (neo-TADs). Hi-C was very sensitive in detecting translocations, and it revealed previously unrecognized complex rearrangements at the breakpoints. In several cases, we observed the formation of fused-TADs promoting ectopic enhancer-promoter interactions that were likely to be involved in the disease pathology. In summary, we show that Hi-C is a sensible method for the detection of complex SVs in a clinical setting. The results help interpret the possible pathogenic effects of the SVs in individuals with DDs.


Assuntos
Cromossomos Humanos/genética , Deficiências do Desenvolvimento/genética , Genoma Humano/genética , Conformação Molecular , Translocação Genética/genética , Montagem e Desmontagem da Cromatina/genética , Pontos de Quebra do Cromossomo , Estudos de Coortes , Humanos , Fatores de Transcrição SOX9/genética , Duplicações Segmentares Genômicas/genética
12.
PLoS One ; 15(5): e0232719, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32392228

RESUMO

BACKGROUND: Atrial fibrillation (AF) is predicted to affect around 17.9 million individuals in Europe by 2060. The disease is associated with severe electrical and structural remodelling of the heart, and increased the risk of stroke and heart failure. In order to improve treatment and find new drug targets, the field needs to better comprehend the exact molecular mechanisms in these remodelling processes. OBJECTIVES: This study aims to identify gene and miRNA networks involved in the remodelling of AF hearts in AF patients with mitral valve regurgitation (MVR). METHODS: Total RNA was extracted from right atrial biopsies from patients undergoing surgery for mitral valve replacement or repair with AF and without history of AF to test for differentially expressed genes and miRNAs using RNA-sequencing and miRNA microarray. In silico predictions were used to construct a mRNA-miRNA network including differentially expressed mRNAs and miRNAs. Gene and chromosome enrichment analysis were used to identify molecular pathways and high-density AF loci. RESULTS: We found 644 genes and 43 miRNAs differentially expressed in AF patients compared to controls. From these lists, we identified 905 pairs of putative miRNA-mRNA interactions, including 37 miRNAs and 295 genes. Of particular note, AF-associated miR-130b-3p, miR-338-5p and miR-208a-3p were differentially expressed in our AF tissue samples. These miRNAs are predicted regulators of several differentially expressed genes associated with cardiac conduction and fibrosis. We identified two high-density AF loci in chromosomes 14q11.2 and 6p21.3. CONCLUSIONS: AF in MVR patients is associated with down-regulation of ion channel genes and up-regulation of extracellular matrix genes. Other AF related genes are dysregulated and several are predicted to be targeted by miRNAs. Our novel miRNA-mRNA regulatory network provides new insights into the mechanisms of AF.


Assuntos
Fibrilação Atrial/genética , Redes Reguladoras de Genes , MicroRNAs/genética , Insuficiência da Valva Mitral/genética , Fibrilação Atrial/complicações , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Biópsia , Estudos de Casos e Controles , Cromossomos Humanos/genética , Epistasia Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Genoma Humano , Átrios do Coração/fisiopatologia , Humanos , Masculino , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/complicações , Insuficiência da Valva Mitral/fisiopatologia , Análise de Componente Principal , Remodelação Vascular
13.
PLoS One ; 15(3): e0229996, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119713

RESUMO

Clinical studies have recently demonstrated that autologous transplantation of mobilized dental pulp stem cells is a safe and efficacious potential therapy for pulp regeneration. However, some limitations need to be addressed, such as the high cost of the safety and quality control tests for isolated individual dental pulp cell products before transplantation. Therefore, more efficient in vitro culturing of human dental pulp stem cells might be useful for providing low cost and high reliability testing for pulp regeneration therapy. In this study, we established a novel immortalized dental pulp stem cell line by co-expressing a mutant cyclin-dependent kinase 4 (CDK4R24C), Cyclin D1, and telomerase reverse transcriptase (TERT). The established cell line maintained its original diploid chromosomes and stemness characteristics and exhibited an enhanced proliferation rate. In addition, we showed the immortalized human dental pulp stem cells still keeps their osteogenic and adipogenic differentiation abilities under appropriate culture conditions even though the cell proliferation was accelerated. Taken together, our established cell lines could serve as a useful in vitro tool for pulp regeneration therapy, and can contribute to reproducibility and ease of cell handling, thereby saving time and costs associated with safety and quality control tests.


Assuntos
Ciclo Celular/genética , Cromossomos Humanos/genética , Polpa Dentária/citologia , Transplante de Células-Tronco , Células-Tronco/citologia , Proliferação de Células , Senescência Celular , Ciclina D1/genética , Quinase 4 Dependente de Ciclina/genética , Expressão Gênica , Humanos , Telomerase/genética
14.
Appl Immunohistochem Mol Morphol ; 28(2): 123-129, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32044880

RESUMO

Clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) are the 2 most common RCCs. However, some RCCs can have both clear cell and papillary features, including clear cell papillary RCC (ccpRCC). They can be a diagnostic challenge in daily practice. Accurate diagnosis of these tumors is important for both patient prognosis and appropriate treatment. Fourteen RCCs with papillary architecture, clear cytoplasm and low Fuhrman grade were analyzed by SNP-based chromosome microarray (CMA). Seven cases had pathologic features of ccpRCC, and all had normal genomic profiles except one that had copy neutral loss of heterozygosity (cnLOH) of chromosome 3 and loss of one copy of the X chromosome. The remaining 7 cases also had papillae and clear cytoplasm. Two of these cases showed losses of chromosome 3 which are typically found in ccRCC. One had a gain of chromosome 7, which is commonly seen in pRCC. The remaining 4 had no alterations of chromosome 3 or 7. However, 3 of these 4 had monosomy 8, which are consistent with RCC with monosomy 8. The remaining case had no copy number alterations. This study shows that low-grade RCC with papillae and clear cell phenotype represents a heterogeneous group, including ccpRCC, ccRCC, pRCC, and RCC with monosomy 8. CMA analysis can be useful for the differential diagnosis of these neoplasms.


Assuntos
Carcinoma Papilar , Carcinoma de Células Renais , Aberrações Cromossômicas , Cromossomos Humanos/genética , Neoplasias Renais , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Carcinoma Papilar/diagnóstico , Carcinoma Papilar/genética , Carcinoma Papilar/patologia , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Diagnóstico Diferencial , Feminino , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Masculino , Pessoa de Meia-Idade
16.
BMC Genomics ; 21(1): 95, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32000688

RESUMO

BACKGROUND: Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1-1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. RESULTS: Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. CONCLUSIONS: Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure.


Assuntos
Cromatina/química , Cromossomos Humanos/genética , Variação Estrutural do Genoma , Linhagem Celular Tumoral , Cromatina/genética , Montagem e Desmontagem da Cromatina , Cromossomos Humanos/química , Elementos Facilitadores Genéticos , Humanos , Modelos Lineares , Deleção de Sequência , Inversão de Sequência
17.
Nat Commun ; 11(1): 877, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054837

RESUMO

Epstein-Barr virus (EBV) genomes persist in latently infected cells as extrachromosomal episomes that attach to host chromosomes through the tethering functions of EBNA1, a viral encoded sequence-specific DNA binding protein. Here we employ circular chromosome conformation capture (4C) analysis to identify genome-wide associations between EBV episomes and host chromosomes. We find that EBV episomes in Burkitt's lymphoma cells preferentially associate with cellular genomic sites containing EBNA1 binding sites enriched with B-cell factors EBF1 and RBP-jK, the repressive histone mark H3K9me3, and AT-rich flanking sequence. These attachment sites correspond to transcriptionally silenced genes with GO enrichment for neuronal function and protein kinase A pathways. Depletion of EBNA1 leads to a transcriptional de-repression of silenced genes and reduction in H3K9me3. EBV attachment sites in lymphoblastoid cells with different latency type show different correlations, suggesting that host chromosome attachment sites are functionally linked to latency type gene expression programs.


Assuntos
Sítios de Ligação Microbiológicos/genética , Sítios de Ligação Microbiológicos/fisiologia , Herpesvirus Humano 4/genética , Herpesvirus Humano 4/fisiologia , Interações entre Hospedeiro e Microrganismos/genética , Linfoma de Burkitt/genética , Linfoma de Burkitt/virologia , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Cromossomos Humanos/virologia , Epigênese Genética , Antígenos Nucleares do Vírus Epstein-Barr/fisiologia , Herpesvirus Humano 4/patogenicidade , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Modelos Biológicos , Plasmídeos/genética , Latência Viral/genética , Latência Viral/fisiologia
18.
Nat Biotechnol ; 38(4): 433-438, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32042167

RESUMO

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1-4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 µg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sequenciamento por Nanoporos/métodos , RNA Guia/metabolismo , Animais , Células Cultivadas , Cromossomos Humanos/genética , Loci Gênicos/genética , Variação Genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Análise de Sequência de DNA
19.
Hum Genet ; 139(4): 531-543, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32030560

RESUMO

We present a comprehensive clinically oriented workflow for large-insert genome sequencing (liGS)-based nucleotide level resolution and interpretation of de novo (dn) apparently balanced chromosomal abnormalities (BCA) in prenatal diagnosis (PND). Retrospective or concomitant with conventional PND and liGS, molecular and newly developed clinically inspired bioinformatic tools (TAD-GConTool and CNV-ConTool) are applied to analyze and assess the functional and phenotypic outcome of dn structural variants (dnSVs). Retrospective analysis of four phenotype-associated dnSVs identified during conventional PND precisely reveal the genomic elements disrupted by the translocation breakpoints. Identification of autosomal dominant disease due to the disruption of ANKS1B and WDR26 by t(12;17)(q23.1;q21.33)dn and t(1;3)(q24.11;p25.3)dn breakpoints, respectively, substantiated the proposed workflow. We then applied this workflow to two ongoing prenatal cases with apparently balanced dnBCAs: 46,XX,t(16;17)(q24;q21.3)dn referred for increased risk on combined first trimester screening and 46,XY,t(2;19)(p13;q13.1)dn referred due to a previous trisomy 21 pregnancy. Translocation breakpoints in the t(16;17) involve ANKRD11 and WNT3 and disruption of ANKRD11 resulted in KBG syndrome confirmed in postnatal follow-up. Breakpoints in the t(2;19) are within ATP6V1B1 and the 3' UTR of CEP89, and are not interpreted to cause disease. Genotype-phenotype correlation confirms the causative role of WDR26 in the Skraban-Deardorff and 1q41q42 microdeletion phenocopy syndromes, and that disruption of ANKS1B causes ANKS1B haploinsufficiency syndrome. In sum, we show that an liGS-based approach can be realized in PND care providing additional information concerning clinical outcomes of dnBCAs in patients with such rearrangements.


Assuntos
Anormalidades Múltiplas , Doenças do Desenvolvimento Ósseo , Transtornos Cromossômicos , Cromossomos Humanos/genética , Facies , Genes Dominantes , Deficiência Intelectual , Diagnóstico Pré-Natal , Anormalidades Dentárias , Translocação Genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Adolescente , Adulto , Doenças do Desenvolvimento Ósseo/diagnóstico , Doenças do Desenvolvimento Ósseo/genética , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Feminino , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Masculino , Gravidez , Anormalidades Dentárias/diagnóstico , Anormalidades Dentárias/genética , Fluxo de Trabalho
20.
Methods Mol Biol ; 2119: 165-181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989524

RESUMO

Chromosome-derived extrachromosomal circular DNA elements (eccDNAs) are detected in all eukaryotes examined so far. Here I describe the Circle-Seq protocol, applicable for physical enrichment of eccDNAs of a broad size range, combined with sequence confirmation of circular structures.Briefly, by concise alkaline treatment and gentle gravity flow-through an ion-exchange column, eccDNAs are enriched in the eluate fraction. EccDNAs are enzymatically isolated by extensive Plasmid-Safe DNase digestion of linear chromosomes and further enriched by φ29 rolling circle amplification. By means of high throughput sequencing of amplified eccDNA and custom eccDNA mapping software, around ten-thousand unique eccDNA types could be detected at nucleotide resolution in a million human muscle nuclei by this method.


Assuntos
Cromossomos Humanos , DNA Circular , Análise de Sequência de DNA , Animais , Linhagem Celular , Cromatografia por Troca Iônica , Cromossomos Humanos/química , Cromossomos Humanos/genética , DNA Circular/química , DNA Circular/genética , DNA Circular/isolamento & purificação , Humanos
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