RESUMO
Durable antibody immunity depends on long-lived plasma cells (LLPCs) that primarily reside in the bone marrow (BM). However, due to LLPC rarity, it has not been possible to define their phenotypes or determine their heterogeneity. By single-cell mRNA sequencing, cytometry and a genetic pulse-chase mouse model, we show that IgG and IgM LLPCs display an EpCAMhiCXCR3- phenotype, whereas IgA LLPCs are Ly6AhiTigit-. While IgG and IgA LLPCs are mainly contributed by somatically hypermutated cells following immunization or infection, cells with innate properties and public antibodies are found in IgA and IgM LLPC compartments. Particularly, IgM LLPCs are highly enriched with public clones shared among different individual animals, differentiated in a T cell-independent manner and have affinity for self-antigens and microbial-derived antigens. Taken together, our work reveals different routes toward LLPC development and paves the way for deeper understanding of cellular and molecular underpinnings of long-term antibody immunity.
Assuntos
Microbiota , Plasmócitos , Camundongos , Animais , Autoantígenos , Imunização , Imunoglobulina M , Imunoglobulina A , Imunoglobulina GRESUMO
Analyzing the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from clinical samples is crucial for understanding viral spread and evolution as well as for vaccine development. Existing RNA sequencing methods are demanding on user technique and time and, thus, not ideal for time-sensitive clinical samples; these methods are also not optimized for high performance on viral genomes. We developed a facile, practical, and robust approach for metagenomic and deep viral sequencing from clinical samples. We demonstrate the utility of our approach on pharyngeal, sputum, and stool samples collected from coronavirus disease 2019 (COVID-19) patients, successfully obtaining whole metatranscriptomes and complete high-depth, high-coverage SARS-CoV-2 genomes with high yield and robustness. With a shortened hands-on time from sample to virus-enriched sequencing-ready library, this rapid, versatile, and clinic-friendly approach will facilitate molecular epidemiology studies during current and future outbreaks.
Assuntos
COVID-19/genética , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética , SARS-CoV-2/genética , Sequenciamento Completo do Genoma , Animais , Humanos , Camundongos , Células NIH 3T3 , RNA Viral/metabolismo , SARS-CoV-2/metabolismoRESUMO
Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.
Assuntos
Taxa de Mutação , Análise de Célula Única , Espermatozoides/metabolismo , Adulto , Conversão Gênica , Estudo de Associação Genômica Ampla , Instabilidade Genômica , Humanos , Masculino , Meiose , Técnicas Analíticas Microfluídicas , Recombinação Genética , Espermatozoides/citologiaRESUMO
Antibody affinity maturation depends on positive selection in germinal centres (GCs) of rare B cell clones that acquire higher-affinity B cell receptors via somatic hypermutation, present more antigen to follicular helper T (TFH) cells and, consequently, receive more contact-dependent T cell help1. As these GC B cells and TFH cells do not maintain long-lasting contacts in the chaotic GC environment2-4, it is unclear how sufficient T cell help is cumulatively focused onto those rare clones. Here we show that, upon stimulation of CD40, GC B cells upregulate the chemokine CCL22 and to a lesser extent CCL17. By engaging the chemokine receptor CCR4 on TFH cells, CCL22 and CCL17 can attract multiple helper cells from a distance, thus increasing the chance of productive help. During a GC response, B cells that acquire higher antigen-binding affinities express higher levels of CCL22, which in turn 'highlight' these high-affinity GC B cells. Acute increase or blockade of TFH cells helps to rapidly increase or decrease CCL22 expression by GC B cells, respectively. Therefore, a chemokine-based intercellular reaction circuit links the amount of T cell help that individual B cells have received recently to their subsequent ability to attract more help. When CCL22 and CCL17 are ablated in B cells, GCs form but B cells are not affinity-matured efficiently. When competing with wild-type B cells in the same reaction, B cells lacking CCL22 and CCL17 receive less T cell help to maintain GC participation or develop into bone-marrow plasma cells. By uncovering a chemokine-mediated mechanism that highlights affinity-improved B cells for preferential help from TFH cells, our study reveals a principle of spatiotemporal orchestration of GC positive selection.
Assuntos
Quimiocina CCL22/metabolismo , Centro Germinativo/citologia , Centro Germinativo/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Células Cultivadas , Quimiocina CCL17/deficiência , Quimiocina CCL17/genética , Quimiocina CCL22/deficiência , Quimiocina CCL22/genética , Feminino , Humanos , Masculino , Camundongos , Tonsila Palatina/citologia , Receptores CCR4/deficiência , Receptores CCR4/genética , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Regulação para CimaRESUMO
Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.
Assuntos
Células Clonais/metabolismo , Saúde , Mutagênese , Mutação , Especificidade de Órgãos , Idoso de 80 Anos ou mais , Biópsia , Cadáver , Cárdia/metabolismo , Proliferação de Células , Células Clonais/citologia , Esôfago/metabolismo , Feminino , Genômica , Humanos , MasculinoRESUMO
Since its establishment in 2009, single-cell RNA sequencing (RNA-seq) has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. Although most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, with the same cell line and bioinformatics pipeline, we developed directly comparable datasets for each of three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. Although each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.
Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Técnicas Analíticas Microfluídicas , RNA/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcriptoma , Automação Laboratorial , Sequência de Bases , Linhagem Celular , Biologia Computacional , Análise Custo-Benefício , Código de Barras de DNA Taxonômico , Perfilação da Expressão Gênica/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Técnicas Analíticas Microfluídicas/economia , Reprodutibilidade dos Testes , Análise de Sequência de RNA/economia , Análise de Célula Única/economia , Fluxo de TrabalhoRESUMO
Humoral immune responses to immunization and infection and susceptibilities to antibody-mediated autoimmunity are generally lower in males1-3. However, the mechanisms underlying such sexual dimorphism are not well understood. Here we show that there are intrinsic differences between the B cells that produce germinal centres in male and female mice. We find that antigen-activated male B cells do not position themselves as efficiently as female B cells in the centre of follicles in secondary lymphoid organs, in which germinal centres normally develop. Moreover, GPR174-an X-chromosome-encoded G-protein-coupled receptor-suppresses the formation of germinal centres in male, but not female, mice. This effect is intrinsic to B cells, and correlates with the GPR174-enhanced positioning of B cells towards the T-cell-B-cell border of follicles, and the distraction of male, but not female, B cells from S1PR2-driven follicle-centre localization. Biochemical fractionation of conditioned media that induce B-cell migration in a GPR174-dependent manner identifies CCL21 as a GPR174 ligand. In response to CCL21, GPR174 triggers a calcium flux and preferentially induces the migration of male B cells; GPR174 also becomes associated with more Gαi protein in male than in female B cells. Male B cells from orchidectomized mice exhibit impaired GPR174-mediated migration to CCL21, and testosterone treatment rescues this defect. Female B cells from testosterone-treated mice exhibit male-like GPR174-Gαi association and GPR174-mediated migration. Deleting GPR174 from male B cells causes more efficient positioning towards the follicular centre, the formation of more germinal centres and an increased susceptibility to B-cell-dependent experimental autoimmune encephalomyelitis. By identifying GPR174 as a receptor for CCL21 and demonstrating its sex-dependent control of B-cell positioning and participation in germinal centres, we have revealed a mechanism by which B-cell physiology is fine-tuned to impart sexual dimorphism to humoral immunity.
Assuntos
Quimiocina CCL21/imunologia , Imunidade Humoral , Receptores Acoplados a Proteínas G/imunologia , Caracteres Sexuais , Animais , Linfócitos B/imunologia , Movimento Celular , Células Cultivadas , Quimiocina CCL21/genética , Suscetibilidade a Doenças , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/genéticaRESUMO
Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.
RESUMO
Spatial transcriptomics technology has revolutionized our understanding of cell types and tissue organization, opening possibilities for researchers to explore transcript distributions at subcellular levels. However, existing methods have limitations in resolution, sensitivity, or speed. To overcome these challenges, we introduce SPRINTseq (Spatially Resolved and signal-diluted Next-generation Targeted sequencing), an innovative in situ sequencing strategy that combines hybrid block coding and molecular dilution strategies. Our method enables fast and sensitive high-resolution data acquisition, as demonstrated by recovering over 142 million transcripts using a 108-gene panel from 453,843 cells from four mouse brain coronal slices in less than 2 d. Using this advanced technology, we uncover the cellular and subcellular molecular architecture of Alzheimer's disease, providing additional information into abnormal cellular behaviors and their subcellular mRNA distribution. This improved spatial transcriptomics technology holds great promise for exploring complex biological processes and disease mechanisms.
Assuntos
Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Animais , Camundongos , RNA Mensageiro/genética , TranscriptomaRESUMO
BACKGROUND: Pathogenic variants in SCN5A can result in long QT syndrome type 3, a life-threatening genetic disease. Adenine base editors can convert targeted A T base pairs to G C base pairs, offering a promising tool to correct pathogenic variants. METHODS: We generated a long QT syndrome type 3 mouse model by introducing the T1307M pathogenic variant into the Scn5a gene. The adenine base editor was split into 2 smaller parts and delivered into the heart by adeno-associated virus serotype 9 (AAV9-ABEmax) to correct the T1307M pathogenic variant. RESULTS: Both homozygous and heterozygous T1307M mice showed significant QT prolongation. Carbachol administration induced Torsades de Pointes or ventricular tachycardia for homozygous T1307M mice (20%) but not for heterozygous or wild-type mice. A single intraperitoneal injection of AAV9-ABEmax at postnatal day 14 resulted in up to 99.20% Scn5a transcripts corrected in T1307M mice. Scn5a mRNA correction rate >60% eliminated QT prolongation; Scn5a mRNA correction rate <60% alleviated QT prolongation. Partial Scn5a correction resulted in cardiomyocytes heterogeneity, which did not induce severe arrhythmias. We did not detect off-target DNA or RNA editing events in ABEmax-treated mouse hearts. CONCLUSIONS: These findings show that in vivo AAV9-ABEmax editing can correct the variant Scn5a allele, effectively ameliorating arrhythmia phenotypes. Our results offer a proof of concept for the treatment of hereditary arrhythmias.
Assuntos
Doença do Sistema de Condução Cardíaco , Edição de Genes , Síndrome do QT Longo , Camundongos , Animais , Síndrome do QT Longo/genética , Síndrome do QT Longo/terapia , Síndrome do QT Longo/diagnóstico , Arritmias Cardíacas , Miócitos Cardíacos , Adenina , RNA Mensageiro , Canal de Sódio Disparado por Voltagem NAV1.5/genética , MutaçãoRESUMO
Genomic-scale somatic copy number alterations in healthy humans are difficult to investigate because of low occurrence rates and the structural variations' stochastic natures. Using a Tn5-transposase-assisted single-cell whole-genome sequencing method, we sequenced over 20,000 single lymphocytes from 16 individuals. Then, with the scale increased to a few thousand single cells per individual, we found that about 7.5% of the cells had large-size copy number alterations. Trisomy 21 was the most prevalent aneuploid event among all autosomal copy number alterations, whereas monosomy X occurred most frequently in over-30-yr-old females. In the monosomy X single cells from individuals with phased genomes and identified X-inactivation ratios in bulk, the inactive X Chromosomes were lost more often than the active ones.
Assuntos
Variações do Número de Cópias de DNA , Genômica , Aneuploidia , Feminino , Humanos , Linfócitos , Sequenciamento Completo do GenomaRESUMO
In most organisms, the whole genome is maintained throughout the life span. However, exceptions occur in some species where the genome is reduced during development through a process known as programmed DNA elimination (PDE). In the human and pig parasite Ascaris, PDE occurs during the 4 to 16 cell stages of embryogenesis, when germline chromosomes are fragmented and specific DNA sequences are reproducibly lost in all somatic cells. PDE was identified in Ascaris over 120 years ago, but little was known about its molecular details until recently. Genome sequencing revealed that approximately 1,000 germline-expressed genes are eliminated in Ascaris, suggesting PDE is a gene silencing mechanism. All germline chromosome ends are removed and remodeled during PDE. In addition, PDE increases the number of chromosomes in the somatic genome by splitting many germline chromosomes. Comparative genomics indicates that these germline chromosomes arose from fusion events. PDE separates these chromosomes at the fusion sites. These observations indicate that PDE plays a role in chromosome karyotype and evolution. Furthermore, comparative analysis of PDE in other parasitic and free-living nematodes illustrates conserved features of PDE, suggesting it has important biological significance. We summarize what is known about PDE in Ascaris and its relatives. We also discuss other potential functions, mechanisms, and the evolution of PDE in these parasites of humans and animals of veterinary importance.
Assuntos
Ascaris , Nematoides , Humanos , Animais , Suínos , Ascaris/genética , Cromossomos , Nematoides/genética , Sequência de Bases , DNARESUMO
In females, the pathophysiological mechanism of poor ovarian response (POR) is not fully understood. Considering the expression level of p62 was significantly reduced in the granulosa cells (GCs) of POR patients, this study focused on identifying the role of the selective autophagy receptor p62 in conducting the effect of follicle-stimulating hormone (FSH) on antral follicles (AFs) formation in female mice. The results showed that p62 in GCs was FSH responsive and that its level increased to a peak and then decreased time-dependently either in ovaries or in GCs after gonadotropin induction in vivo. GC-specific deletion of p62 resulted in subfertility, a significantly reduced number of AFs and irregular estrous cycles, which were same as pathophysiological symptom of POR. By conducting mass spectrum analysis, we found the ubiquitination of proteins was decreased, and autophagic flux was blocked in GCs. Specifically, the level of nonubiquitinated Wilms tumor 1 homolog (WT1), a transcription factor and negative controller of GC differentiation, increased steadily. Co-IP results showed that p62 deletion increased the level of ubiquitin-specific peptidase 5 (USP5), which blocked the ubiquitination of WT1. Furthermore, a joint analysis of RNA-seq and the spatial transcriptome sequencing data showed the expression of steroid metabolic genes and FSH receptors pivotal for GCs differentiation decreased unanimously. Accordingly, the accumulation of WT1 in GCs deficient of p62 decreased steroid hormone levels and reduced FSH responsiveness, while the availability of p62 in GCs simultaneously ensured the degradation of WT1 through the ubiquitinâproteasome system and autophagolysosomal system. Therefore, p62 in GCs participates in GC differentiation and AF formation in FSH induction by dynamically controlling the degradation of WT1. The findings of the study contributes to further study the pathology of POR.
Assuntos
Hormônio Foliculoestimulante , Células da Granulosa , Folículo Ovariano , Proteína Sequestossoma-1 , Ubiquitinação , Proteínas WT1 , Animais , Hormônio Foliculoestimulante/metabolismo , Hormônio Foliculoestimulante/farmacologia , Feminino , Proteínas WT1/metabolismo , Proteínas WT1/genética , Camundongos , Folículo Ovariano/metabolismo , Folículo Ovariano/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , Proteína Sequestossoma-1/genética , Camundongos Endogâmicos C57BL , Autofagia/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Humanos , Camundongos KnockoutRESUMO
The primordial to primary follicle transition (PPT) in the ovary is critical to maintain sustainable reproductive resources in female mammals. However, it is unclear how granulosa cells (GCs) of the primary follicle participate in regulating PPT. This study focused on exploring the role of transcription factor Sp1 (SP1) in regulating PPT based on the fact that SP1 is pivotal for pregranulosa cell proliferation before primordial follicle formation. The results showed that mice fertility was prolonged when Sp1 was specifically depleted from GCs (GC- Sp1 -/- ). Besides, the PPT in GC- Sp1 -/- mice was reduced, resulting in more primordial follicles being preserved. Single-cell RNA-seq also indicated that the level of cholesterol metabolism was downregulated in GC- Sp1 -/- mice. Additionally, the PPT was promoted by either overexpression of ferredoxin-1 (FDX1), one of the key genes in mediating cholesterol metabolism or supplementing cholesterol for cultured fetal ovaries. Collectively, SP1 in GCs participates in the metabolism of cholesterol partially by regulating the transcription of Fdx1 during the PPT.
Assuntos
Células da Granulosa , Folículo Ovariano , Feminino , Camundongos , Animais , Folículo Ovariano/metabolismo , Células da Granulosa/metabolismo , Ovário/metabolismo , Mamíferos , Metabolismo dos LipídeosRESUMO
Wireless sensor networks (WSNs) are gaining traction in the realm of network communication, renowned for their adaptability, configuration, and flexibility. The forthcoming network traffic within WSNs can be forecasted through temporal sequence models. In this correspondence, we present a method (TSENet) that can accurately predict the traffic in the cellular network. TSENet is composed of transformers and self-attention network. We have designed a temporal transformer module specifically for extracting temporal features. This module accomplishes this by modeling the traffic flow within each grid of the communication network at both near-term and periodical intervals. Simultaneously, we amalgamate the spatial features of each grid with information from its correlated grids, generating spatial predictions within the spatial transformer. Furthermore, we employ self-attention aggregation to capture dependencies between external factor features and cellular data features. Empirical assessments performed on a genuine cellular traffic dataset offer compelling evidence substantiating the efficacy of TSENet.
RESUMO
Pholidota cantonensis Rolfe is an edible medicinal plant in the genus Pholidota of the family Orchidaceae. This plant is used to prepare medicated food in China and has been reported to possess anti-α-glucosidase activity. To date, little is known about the active substances responsible for the observed anti-α-glucosidase activity. In the present study, we aimed to screen and characterize the α-glucosidase inhibitory fraction of P. cantonensis using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) analysis and molecular docking. As a result, the 50% ethanol fraction obtained from D101 macroporous adsorption resin column chromatography (D50 fraction) had the highest total phenol content (353.83 ± 6.06 mg GAE/g) and the most prominent α-glucosidase inhibitory activity (IC50 = 30.01 ± 7.30 µg/mL). Forty-five compounds were identified from the D50 fraction by using UPLC-Q-TOF-MS/MS analysis. Molecular docking results showed that six main constituents, namely, crepidatin, 2,7-dihydroxy-4-methoxyl-9,10-dihydrophenylene, 4,4',5,6-tetrahydroxystilbene, 4,7-dihydroxy-2-methoxyl-9,10-dihydrophenylene, (-)-lariciresinol, and thunalbene, in the D50 fraction occupied the catalytic sites of α-glucosidase through strong hydrophobic interactions, hydrogen bonding, and other patterns. The binding energies were between - 29.95 and - 11.41 kJ/mol, indicating good binding between the tested compounds and α-glucosidase. The active ingredients responsible for the α-glucosidase inhibitory activity may include phenanthrenes, stilbenes, dibenzyls, and lignans. The D50 fraction has potential value for developing innovative drugs for the prevention and treatment of diabetes mellitus (DM) and is worthy of in-depth research.
RESUMO
Alzheimer's Disease (AD) is a progressive neurodegenerative disorder. Due to the subtlety of symptoms in the early stages of AD, rapid and accurate clinical diagnosis is challenging, leading to a high rate of misdiagnosis. Current research on early diagnosis of AD has not sufficiently focused on tracking the progression of the disease over an extended period in subjects. To address this issue, this paper proposes an ensemble model for assisting early diagnosis of AD that combines structural magnetic resonance imaging (sMRI) data from two time points with clinical information. The model employs a three-dimensional convolutional neural network (3DCNN) and twin neural network modules to extract features from the sMRI data of subjects at two time points, while a multi-layer perceptron (MLP) is used to model the clinical information of the subjects. The objective is to extract AD-related features from the multi-modal data of the subjects as much as possible, thereby enhancing the diagnostic performance of the ensemble model. Experimental results show that based on this model, the classification accuracy rate is 89% for differentiating AD patients from normal controls (NC), 88% for differentiating mild cognitive impairment converting to AD (MCIc) from NC, and 69% for distinguishing non-converting mild cognitive impairment (MCInc) from MCIc, confirming the effectiveness and efficiency of the proposed method for early diagnosis of AD, as well as its potential to play a supportive role in the clinical diagnosis of early Alzheimer's disease.
Assuntos
Doença de Alzheimer , Diagnóstico Precoce , Imageamento por Ressonância Magnética , Redes Neurais de Computação , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/diagnóstico , Humanos , Imageamento por Ressonância Magnética/métodos , Progressão da Doença , AlgoritmosRESUMO
BACKGROUND: Visualizing genome coverage is of vital importance to inspect and interpret various next-generation sequencing (NGS) data. Besides genome coverage, genome annotations are also crucial in the visualization. While different NGS data require different annotations, how to visualize genome coverage and add the annotations appropriately and conveniently is challenging. Many tools have been developed to address this issue. However, existing tools are often inflexible, complicated, lack necessary preprocessing steps and annotations, and the figures generated support limited customization. RESULTS: Here, we introduce ggcoverage, an R package to visualize and annotate genome coverage of multi-groups and multi-omics. The input files for ggcoverage can be in BAM, BigWig, BedGraph and TSV formats. For better usability, ggcoverage provides reliable and efficient ways to perform read normalization, consensus peaks generation and track data loading with state-of-the-art tools. ggcoverage provides various available annotations to adapt to different NGS data (e.g. WGS/WES, RNA-seq, ChIP-seq) and all the available annotations can be easily superimposed with ' + '. ggcoverage can generate publication-quality plots and users can customize the plots with ggplot2. In addition, ggcoverage supports the visualization and annotation of protein coverage. CONCLUSIONS: ggcoverage provides a flexible, programmable, efficient and user-friendly way to visualize and annotate genome coverage of multi-groups and multi-omics. The ggcoverage package is available at https://github.com/showteeth/ggcoverage under the MIT license, and the vignettes are available at https://showteeth.github.io/ggcoverage/ .
Assuntos
Genoma , Software , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Cromatina por Imunoprecipitação , RNA-SeqRESUMO
The realization of the vast potential of digital PCR (dPCR) to provide extremely accurate and sensitive measurements in the clinical setting has thus far been hindered by challenges such as assay robustness and high costs. Here we introduce a lossless and contamination-free dPCR technology, termed CLEAR-dPCR, which addresses these challenges by completing the dPCR sample preparation, PCR, and readout all in one tube. Optical clearing of the droplet dPCR emulsion was combined with emerging light-sheet fluorescence microscopy, to acquire a three-dimensional (3D) image of a half million droplets sealed in a tube in seconds. CLEAR-dPCR provides ultrahigh-throughput readout results in situ and fundamentally eliminates the possibility of either sample loss or contamination. This approach exhibits improved accuracy over existing dPCR platforms and enables a greatly increased dynamic range to be comparable to that of real-time quantitative PCR.