RESUMO
Welch et al. and Stuart et al. present novel techniques for the integration of single-cell RNA-seq datasets across multiple platforms, individuals, and species. They both extend these strategies to map cell types between RNA-seq datasets with epigenetic properties and in situ transcript profiling. The ability to transfer information between datasets and spatial methods will enable more comprehensive profiling and comparisons of cell populations in complex biological systems.
Assuntos
Perfilação da Expressão Gênica , Genômica , Epigenômica , HumanosRESUMO
Conventional methods for single-cell genome sequencing are limited with respect to uniformity and throughput. Here, we describe sci-L3, a single-cell sequencing method that combines combinatorial indexing (sci-) and linear (L) amplification. The sci-L3 method adopts a 3-level (3) indexing scheme that minimizes amplification biases while enabling exponential gains in throughput. We demonstrate the generalizability of sci-L3 with proof-of-concept demonstrations of single-cell whole-genome sequencing (sci-L3-WGS), targeted sequencing (sci-L3-target-seq), and a co-assay of the genome and transcriptome (sci-L3-RNA/DNA). We apply sci-L3-WGS to profile the genomes of >10,000 sperm and sperm precursors from F1 hybrid mice, mapping 86,786 crossovers and characterizing rare chromosome mis-segregation events in meiosis, including instances of whole-genome equational chromosome segregation. We anticipate that sci-L3 assays can be applied to fully characterize recombination landscapes, to couple CRISPR perturbations and measurements of genome stability, and to other goals requiring high-throughput, high-coverage single-cell sequencing.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas de Amplificação de Ácido Nucleico , Análise de Sequência de DNA , Análise de Sequência de RNA , Análise de Célula Única/métodos , Sequenciamento Completo do Genoma , Animais , Segregação de Cromossomos , Masculino , Meiose/genética , Camundongos , Estudo de Prova de Conceito , Espermatozoides/fisiologia , Transcriptoma , Fluxo de TrabalhoRESUMO
The Healthy Oregon Project (HOP) is a statewide effort that aims to build a large research repository and influence the health of Oregonians through providing no-cost genetic screening to participants for a next-generation sequencing 32-gene panel comprising genes related to inherited cancers and familial hypercholesterolemia. This type of unbiased population screening can detect at-risk individuals who may otherwise be missed by conventional medical approaches. However, challenges exist for this type of high-throughput testing in an academic setting, including developing a low-cost high-efficiency test and scaling up the clinical laboratory for processing large numbers of samples. Modifications to our academic clinical laboratory including efficient test design, robotics, and a streamlined analysis approach increased our ability to test more than 1,000 samples per month for HOP using only one dedicated HOP laboratory technologist. Additionally, enrollment using a HIPAA-compliant smartphone app and sample collection using mouthwash increased efficiency and reduced cost. Here, we present our experience three years into HOP and discuss the lessons learned, including our successes, challenges, opportunities, and future directions, as well as the genetic screening results for the first 13,670 participants tested. Overall, we have identified 730 pathogenic/likely pathogenic variants in 710 participants in 24 of the 32 genes on the panel. The carrier rate for pathogenic/likely pathogenic variants in the inherited cancer genes on the panel for an unselected population was 5.0% and for familial hypercholesterolemia was 0.3%. Our laboratory experience described here may provide a useful model for population screening projects in other states.
Assuntos
Hiperlipoproteinemia Tipo II , Neoplasias , Humanos , Oregon/epidemiologia , Detecção Precoce de Câncer , Testes Genéticos , Hiperlipoproteinemia Tipo II/diagnóstico , Hiperlipoproteinemia Tipo II/epidemiologia , Hiperlipoproteinemia Tipo II/genética , Neoplasias/diagnóstico , Neoplasias/epidemiologia , Neoplasias/genéticaRESUMO
Here we present advancements in single-cell combinatorial indexed Assay for Transposase Accessible Chromatin (sciATAC) to measure chromatin accessibility that leverage nanowell chips to achieve atlas-scale cell throughput (>105 cells) at low cost. The platform leverages the core of the sciATAC workflow where multiple indexed tagmentation reactions are performed, followed by pooling and distribution to a second set of reaction wells for polymerase chain reaction (PCR)-based indexing. In this work, we instead leverage a chip containing 5184 nanowells at the PCR stage of indexing, enabling a 52-fold improvement in scale and reduction in per-cell preparation costs. We detail three variants that balance cell throughput and depth of coverage, and apply these methods to banked mouse brain tissue, producing maps of cell types as well as neuronal subtypes that include integration with existing single-cell Assay for Transposase Accessible Chromatin (scATAC) and scRNA-seq data sets. Our optimized workflow achieves a high fraction of reads that fall within called peaks (>80%) and low cell doublet rates. The high cell coverage technique produces high unique reads per cell, while retaining high enrichment for open chromatin regions, enabling the assessment of >70,000 unique accessible loci on average for each cell profiled. When compared to current methods in the field, our technique provides similar or superior per-cell information with very low levels of cell-to-cell cross talk, and achieves this at a cost point much lower than existing assays.
Assuntos
Cromatina , Transposases , Camundongos , Animais , Transposases/metabolismo , Neurônios/metabolismo , Epigenômica/métodos , Análise de Célula Única/métodosRESUMO
ABSTRACT: Identifying and targeting microenvironment-driven pathways that are active across acute myeloid leukemia (AML) genetic subtypes should allow the development of more broadly effective therapies. The proinflammatory cytokine interleukin-1ß (IL-1ß) is abundant in the AML microenvironment and promotes leukemic growth. Through RNA-sequencing analysis, we identify that IL-1ß-upregulated ASF1B (antisilencing function-1B), a histone chaperone, in AML progenitors compared with healthy progenitors. ASF1B, along with its paralogous protein ASF1A, recruits H3-H4 histones onto the replication fork during S-phase, a process regulated by Tousled-like kinase 1 and 2 (TLKs). Although ASF1s and TLKs are known to be overexpressed in multiple solid tumors and associated with poor prognosis, their functional roles in hematopoiesis and inflammation-driven leukemia remain unexplored. In this study, we identify that ASF1s and TLKs are overexpressed in multiple genetic subtypes of AML. We demonstrate that depletion of ASF1s significantly reduces leukemic cell growth in both in vitro and in vivo models using human cells. Using a murine model, we show that overexpression of ASF1B accelerates leukemia progression. Moreover, Asf1b or Tlk2 deletion delayed leukemia progression, whereas these proteins are dispensable for normal hematopoiesis. Through proteomics and phosphoproteomics analyses, we uncover that the TLK-ASF1 pathway promotes leukemogenesis by affecting the cell cycle and DNA damage pathways. Collectively, our findings identify the TLK1-ASF1 pathway as a novel mediator of inflammatory signaling and a promising therapeutic target for AML treatment across diverse genetic subtypes. Selective inhibition of this pathway offers potential opportunities to intervene effectively, address intratumoral heterogeneity, and ultimately improve clinical outcomes in AML.
Assuntos
Proteínas de Ciclo Celular , Progressão da Doença , Interleucina-1beta , Leucemia Mieloide Aguda , Proteínas Serina-Treonina Quinases , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/genética , Humanos , Animais , Camundongos , Interleucina-1beta/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/genética , Histonas/metabolismo , Histonas/genética , Linhagem Celular Tumoral , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genéticaRESUMO
Linking regulatory DNA elements to their target genes, which may be located hundreds of kilobases away, remains challenging. Here, we introduce Cicero, an algorithm that identifies co-accessible pairs of DNA elements using single-cell chromatin accessibility data and so connects regulatory elements to their putative target genes. We apply Cicero to investigate how dynamically accessible elements orchestrate gene regulation in differentiating myoblasts. Groups of Cicero-linked regulatory elements meet criteria of "chromatin hubs"-they are enriched for physical proximity, interact with a common set of transcription factors, and undergo coordinated changes in histone marks that are predictive of changes in gene expression. Pseudotemporal analysis revealed that most DNA elements remain in chromatin hubs throughout differentiation. A subset of elements bound by MYOD1 in myoblasts exhibit early opening in a PBX1- and MEIS1-dependent manner. Our strategy can be applied to dissect the architecture, sequence determinants, and mechanisms of cis-regulation on a genome-wide scale.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Cromatina/genética , DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Adolescente , Diferenciação Celular/genética , Feminino , Genes Homeobox/genética , Histonas/genética , Humanos , Mioblastos/fisiologia , Fatores de Transcrição/genéticaRESUMO
The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.
Assuntos
Poro Nuclear/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitógenos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-myc/química , Serina/metabolismo , Cicatrização , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
It has been just over 10 years since the initial description of transposase-based methods to prepare high-throughput sequencing libraries, or "tagmentation," in which a hyperactive transposase is used to simultaneously fragment target DNA and append universal adapter sequences. Tagmentation effectively replaced a series of processing steps in traditional workflows with one single reaction. It is the simplicity, coupled with the high efficiency of tagmentation, that has made it a favored means of sequencing library construction and fueled a diverse range of adaptations to assay a variety of molecular properties. In recent years, this has been centered in the single-cell space with a catalog of tagmentation-based assays that have been developed, covering a substantial swath of the regulatory landscape. To date, there have been a number of excellent reviews on single-cell technologies structured around the molecular properties that can be profiled. This review is instead framed around the central components and properties of tagmentation and how they have enabled the development of innovative molecular tools to probe the regulatory landscape of single cells. Furthermore, the granular specifics on cell throughput or richness of data provided by the extensive list of individual technologies are not discussed. Such metrics are rapidly changing and highly sample specific and are better left to studies that directly compare technologies for assays against one another in a rigorously controlled framework. The hope for this review is that, in laying out the diversity of molecular techniques at each stage of these assay platforms, new ideas may arise for others to pursue that will further advance the field of single-cell genomics.
Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Célula Única , Transposases , DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transposases/genéticaRESUMO
T-Box Brain Transcription Factor 1 (TBR1) plays essential roles in brain development, mediating neuronal migration, fate specification, and axon tract formation. While heterozygous loss-of-function and missense TBR1 mutations are associated with neurodevelopmental conditions, the effects of these heterogeneous mutations on brain development have yet to be fully explored. We characterized multiple mouse lines carrying Tbr1 mutations differing by type and exonic location, including the previously generated Tbr1 exon 2-3 knock-out (KO) line, and we analyzed male and female mice at neonatal and adult stages. The frameshift patient mutation A136PfsX80 (A136fs) caused reduced TBR1 protein in cortex similar to Tbr1 KO, while the missense patient mutation K228E caused significant TBR1 upregulation. Analysis of cortical layer formation found similar defects between KO and A136fs homozygotes in their CUX1+ and CTIP2+ layer positions, while K228E homozygosity produced layering defects distinct from these mutants. Meanwhile, the examination of cortical apoptosis found extensive cell death in KO homozygotes but limited cell death in A136fs or K228E homozygotes. Despite their discordant cortical phenotypes, these Tbr1 mutations produced several congruent phenotypes, including anterior commissure reduction in heterozygotes, which was previously observed in humans with TBR1 mutations. These results indicate that patient-specific Tbr1 mutant mice will be valuable translational models for pinpointing shared and distinct etiologies among patients with TBR1-related developmental conditions.SIGNIFICANCE STATEMENT Mutations of the TBR1 gene increase the likelihood of neurodevelopmental conditions such as intellectual disability and autism. Therefore, the study of TBR1 can offer insights into the biological mechanisms underlying these conditions, which affect millions worldwide. To improve the modeling of TBR1-related conditions over current Tbr1 knock-out mice, we created mouse lines carrying Tbr1 mutations identical to those found in human patients. Mice with one mutant Tbr1 copy show reduced amygdalar connections regardless of mutation type, suggesting a core biomarker for TBR1-related disorders. In mice with two mutant Tbr1 copies, brain phenotypes diverge by mutation type, suggesting differences in Tbr1 gene functionality in different patients. These mouse models will serve as valuable tools for understanding genotype-phenotype relationships among patients with neurodevelopmental conditions.
Assuntos
Proteínas de Ligação a DNA , Neurogênese , Proteínas com Domínio T , Animais , Axônios/metabolismo , Proteínas de Ligação a DNA/genética , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Mutação , Neurogênese/genética , Proteínas com Domínio T/genéticaRESUMO
Aneuploidy that arises during meiosis and/or mitosis is a major contributor to early embryo loss. We previously showed that human preimplantation embryos encapsulate missegregated chromosomes into micronuclei while undergoing cellular fragmentation and that fragments can contain chromosomal material, but the source of this DNA was unknown. Here, we leveraged the use of a nonhuman primate model and single-cell DNA-sequencing (scDNA-seq) to examine the chromosomal content of 471 individual samples comprising 254 blastomeres, 42 polar bodies, and 175 cellular fragments from a large number (N = 50) of disassembled rhesus cleavage-stage embryos. Our analysis revealed that the aneuploidy and micronucleation frequency is conserved between humans and macaques, and that fragments encapsulate whole and/or partial chromosomes lost from blastomeres. Single-cell/fragment genotyping showed that these chromosome-containing cellular fragments (CCFs) can be maternally or paternally derived and display double-stranded DNA breaks. DNA breakage was further indicated by reciprocal subchromosomal losses/gains between blastomeres and large segmental errors primarily detected at the terminal ends of chromosomes. By combining time-lapse imaging with scDNA-seq, we determined that multipolar divisions at the zygote or two-cell stage were associated with CCFs and generated a random mixture of chromosomally normal and abnormal blastomeres with uniparental or biparental origins. Despite frequent chromosome missegregation at the cleavage-stage, we show that CCFs and nondividing aneuploid blastomeres showing extensive DNA damage are prevented from incorporation into blastocysts. These findings suggest that embryos respond to chromosomal errors by encapsulation into micronuclei, elimination via cellular fragmentation, and selection against highly aneuploid blastomeres to overcome chromosome instability during preimplantation development.
Assuntos
Aneuploidia , Blastocisto/citologia , Blastômeros/citologia , Micronúcleos com Defeito Cromossômico/embriologia , Animais , Segregação de Cromossomos , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Feminino , Macaca , Análise de Célula ÚnicaRESUMO
Here we present a comprehensive map of the accessible chromatin landscape of the mouse hippocampus at single-cell resolution. Substantial advances of this work include the optimization of a single-cell combinatorial indexing assay for transposase accessible chromatin (sci-ATAC-seq); a software suite, scitools, for the rapid processing and visualization of single-cell combinatorial indexing data sets; and a valuable resource of hippocampal regulatory networks at single-cell resolution. We used sci-ATAC-seq to produce 2346 high-quality single-cell chromatin accessibility maps with a mean unique read count per cell of 29,201 from both fresh and frozen hippocampi, observing little difference in accessibility patterns between the preparations. By using this data set, we identified eight distinct major clusters of cells representing both neuronal and nonneuronal cell types and characterized the driving regulatory factors and differentially accessible loci that define each cluster. Within pyramidal neurons, we identified four major clusters, including CA1 and CA3 neurons, and three additional subclusters. We then applied a recently described coaccessibility framework, Cicero, which identified 146,818 links between promoters and putative distal regulatory DNA. Identified coaccessibility networks showed cell-type specificity, shedding light on key dynamic loci that reconfigure to specify hippocampal cell lineages. Lastly, we performed an additional sci-ATAC-seq preparation from cultured hippocampal neurons (899 high-quality cells, 43,532 mean unique reads) that revealed substantial alterations in their epigenetic landscape compared with nuclei from hippocampal tissue. This data set and accompanying analysis tools provide a new resource that can guide subsequent studies of the hippocampus.
Assuntos
Cromatina/genética , Hipocampo/metabolismo , Células Piramidais/metabolismo , Animais , Linhagem da Célula/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/metabolismo , Epigenômica/métodos , Camundongos , Plasticidade Neuronal/genética , Células Piramidais/citologia , Análise de Sequência de DNA , Análise de Célula Única/métodos , Transposases/genética , Transposases/metabolismoRESUMO
Aging is a significant risk factor for cardiovascular disease. Despite the fact that endothelial cells play critical roles in cardiovascular function and disease, the molecular impact of aging on this cell population in many organ systems remains unknown. In this study, we sought to determine age-associated transcriptional alterations in cardiac endothelial cells. Highly enriched populations of endothelial cells (ECs) isolated from the heart, brain, and kidney of young (3 mo) and aged (24 mo) C57/BL6 mice were profiled for RNA expression via bulk RNA sequencing. Approximately 700 cardiac endothelial transcripts significantly differ by age. Gene set enrichment analysis indicated similar patterns for cellular pathway perturbations. Receptor-ligand comparisons indicated parallel alterations in age-affected circulating factors and cardiac endothelial-expressed receptors. Gene and pathway enrichment analyses show that age-related transcriptional response of cardiac endothelial cells is distinct from that of endothelial cells derived from the brain or kidney vascular bed. Furthermore, single-cell analysis identified nine distinct EC subtypes and shows that the Apelin Receptor-enriched subtype is reduced with age in mouse heart. Finally, we identify age-dysregulated genes in specific aged cardiac endothelial subtypes.
Assuntos
Envelhecimento/genética , Células Endoteliais/fisiologia , Regulação da Expressão Gênica , Miocárdio/citologia , Molécula 1 de Adesão de Célula Vascular , Animais , Encéfalo/citologia , Células Endoteliais/classificação , Rim/citologia , Masculino , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA , Análise de Célula Única , Molécula 1 de Adesão de Célula Vascular/genéticaRESUMO
Human genomes are diploid and, for their complete description and interpretation, it is necessary not only to discover the variation they contain but also to arrange it onto chromosomal haplotypes. Although whole-genome sequencing is becoming increasingly routine, nearly all such individual genomes are mostly unresolved with respect to haplotype, particularly for rare alleles, which remain poorly resolved by inferential methods. Here, we review emerging technologies for experimentally resolving (that is, 'phasing') haplotypes across individual whole-genome sequences. We also discuss computational methods relevant to their implementation, metrics for assessing their accuracy and completeness, and the relevance of haplotype information to applications of genome sequencing in research and clinical medicine.
Assuntos
Genoma Humano , Haplótipos , Animais , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Desequilíbrio de Ligação , Modelos Genéticos , Análise de Sequência de DNARESUMO
Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.
Assuntos
Adenocarcinoma/genética , Mapeamento Cromossômico/métodos , Variações do Número de Cópias de DNA/genética , Lobo Frontal/citologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Pancreáticas/genética , Análise de Sequência de DNA/métodos , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral , Biblioteca Gênica , Genoma Humano/genética , Células HeLa , Humanos , Macaca mulattaRESUMO
The HeLa cell line was established in 1951 from cervical cancer cells taken from a patient, Henrietta Lacks. This was the first successful attempt to immortalize human-derived cells in vitro. The robust growth and unrestricted distribution of HeLa cells resulted in its broad adoption--both intentionally and through widespread cross-contamination--and for the past 60 years it has served a role analogous to that of a model organism. The cumulative impact of the HeLa cell line on research is demonstrated by its occurrence in more than 74,000 PubMed abstracts (approximately 0.3%). The genomic architecture of HeLa remains largely unexplored beyond its karyotype, partly because like many cancers, its extensive aneuploidy renders such analyses challenging. We carried out haplotype-resolved whole-genome sequencing of the HeLa CCL-2 strain, examined point- and indel-mutation variations, mapped copy-number variations and loss of heterozygosity regions, and phased variants across full chromosome arms. We also investigated variation and copy-number profiles for HeLa S3 and eight additional strains. We find that HeLa is relatively stable in terms of point variation, with few new mutations accumulating after early passaging. Haplotype resolution facilitated reconstruction of an amplified, highly rearranged region of chromosome 8q24.21 at which integration of the human papilloma virus type 18 (HPV-18) genome occurred and that is likely to be the event that initiated tumorigenesis. We combined these maps with RNA-seq and ENCODE Project data sets to phase the HeLa epigenome. This revealed strong, haplotype-specific activation of the proto-oncogene MYC by the integrated HPV-18 genome approximately 500 kilobases upstream, and enabled global analyses of the relationship between gene dosage and expression. These data provide an extensively phased, high-quality reference genome for past and future experiments relying on HeLa, and demonstrate the value of haplotype resolution for characterizing cancer genomes and epigenomes.
Assuntos
Epigenômica , Genoma Humano/genética , Aneuploidia , Variações do Número de Cópias de DNA , Feminino , Genes myc/genética , Haplótipos , Células HeLa , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/fisiologia , Humanos , Dados de Sequência Molecular , Mutação , Proto-Oncogene Mas , Análise de Sequência de DNA , Ativação Transcricional/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologiaRESUMO
We describe a method that exploits contiguity preserving transposase sequencing (CPT-seq) to facilitate the scaffolding of de novo genome assemblies. CPT-seq is an entirely in vitro means of generating libraries comprised of 9216 indexed pools, each of which contains thousands of sparsely sequenced long fragments ranging from 5 kilobases to > 1 megabase. These pools are "subhaploid," in that the lengths of fragments contained in each pool sums to â¼5% to 10% of the full genome. The scaffolding approach described here, termed fragScaff, leverages coincidences between the content of different pools as a source of contiguity information. Specifically, CPT-seq data is mapped to a de novo genome assembly, followed by the identification of pairs of contigs or scaffolds whose ends disproportionately co-occur in the same indexed pools, consistent with true adjacency in the genome. Such candidate "joins" are used to construct a graph, which is then resolved by a minimum spanning tree. As a proof-of-concept, we apply CPT-seq and fragScaff to substantially boost the contiguity of de novo assemblies of the human, mouse, and fly genomes, increasing the scaffold N50 of de novo assemblies by eight- to 57-fold with high accuracy. We also demonstrate that fragScaff is complementary to Hi-C-based contact probability maps, providing midrange contiguity to support robust, accurate chromosome-scale de novo genome assemblies without the need for laborious in vivo cloning steps. Finally, we demonstrate CPT-seq as a means of anchoring unplaced novel human contigs to the reference genome as well as for detecting misassembled sequences.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transposases/metabolismo , Animais , Biologia Computacional/métodos , Biblioteca Gênica , Genômica/métodos , Humanos , Camundongos , SoftwareRESUMO
Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed-Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole-genome sequencing-mediated copy number analysis of purified HRS cells. We performed low-coverage sequencing of small numbers of intact HRS cells and paired non-neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS-derived cell lines (KM-H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD-1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236-4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases.
Assuntos
Doença de Hodgkin/genética , Membro 14 de Receptores do Fator de Necrose Tumoral/genética , Linhagem Celular Tumoral , Separação Celular , Citometria de Fluxo , Doença de Hodgkin/metabolismo , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Membro 14 de Receptores do Fator de Necrose Tumoral/biossíntese , Membro 14 de Receptores do Fator de Necrose Tumoral/deficiência , Células de Reed-SternbergRESUMO
Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e. rate of cleavage at 'off-target' sites), and efficient/facile means for delivery to desired target cells. Here, we describe the development of a single-chain rare-cleaving nuclease architecture, which we designate 'megaTAL', in which the DNA binding region of a transcription activator-like (TAL) effector is used to 'address' a site-specific meganuclease adjacent to a single desired genomic target site. This architecture allows the generation of extremely active and hyper-specific compact nucleases that are compatible with all current viral and nonviral cell delivery methods.
Assuntos
Clivagem do DNA , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Técnicas de Inativação de Genes , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Engenharia Genética , Genômica/métodos , Células HEK293 , Humanos , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
We have adapted transposase-based in vitro shotgun library construction ("tagmentation") for whole-genome bisulfite sequencing. This method, Tn5mC-seq, enables a >100-fold reduction in starting material relative to conventional protocols, such that we generate highly complex bisulfite sequencing libraries from as little as 10 ng of input DNA, and ample useful sequences from 1 ng of input DNA. We demonstrate Tn5mC-seq by sequencing the methylome of a human lymphoblastoid cell line to â¼8.6× high-quality coverage of each strand.