RESUMO
Analyzing the spatial organization of molecules in cells and tissues is a cornerstone of biological research and clinical practice. However, despite enormous progress in molecular profiling of cellular constituents, spatially mapping them remains a disjointed and specialized machinery-intensive process, relying on either light microscopy or direct physical registration. Here, we demonstrate DNA microscopy, a distinct imaging modality for scalable, optics-free mapping of relative biomolecule positions. In DNA microscopy of transcripts, transcript molecules are tagged in situ with randomized nucleotides, labeling each molecule uniquely. A second in situ reaction then amplifies the tagged molecules, concatenates the resulting copies, and adds new randomized nucleotides to uniquely label each concatenation event. An algorithm decodes molecular proximities from these concatenated sequences and infers physical images of the original transcripts at cellular resolution with precise sequence information. Because its imaging power derives entirely from diffusive molecular dynamics, DNA microscopy constitutes a chemically encoded microscopy system.
Assuntos
DNA/química , Microscopia de Fluorescência/métodos , Reação em Cadeia da Polimerase , Algoritmos , Sequência de Bases , Linhagem Celular , Difusão Facilitada/genética , Feminino , Corantes Fluorescentes/química , Humanos , Nucleotídeos/química , Fótons , Coloração e Rotulagem/métodosRESUMO
Metagenomic sequencing is revolutionizing the detection and characterization of microbial species, and a wide variety of software tools are available to perform taxonomic classification of these data. The fast pace of development of these tools and the complexity of metagenomic data make it important that researchers are able to benchmark their performance. Here, we review current approaches for metagenomic analysis and evaluate the performance of 20 metagenomic classifiers using simulated and experimental datasets. We describe the key metrics used to assess performance, offer a framework for the comparison of additional classifiers, and discuss the future of metagenomic data analysis.
Assuntos
Bactérias/classificação , Benchmarking/métodos , Fungos/classificação , Metagenoma/genética , Metagenômica/métodos , Vírus/classificação , Bactérias/genética , Bases de Dados Genéticas , Fungos/genética , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Software , Vírus/genéticaRESUMO
Somatic LINE-1 (L1) retrotransposition during neurogenesis is a potential source of genotypic variation among neurons. As a neurogenic niche, the hippocampus supports pronounced L1 activity. However, the basal parameters and biological impact of L1-driven mosaicism remain unclear. Here, we performed single-cell retrotransposon capture sequencing (RC-seq) on individual human hippocampal neurons and glia, as well as cortical neurons. An estimated 13.7 somatic L1 insertions occurred per hippocampal neuron and carried the sequence hallmarks of target-primed reverse transcription. Notably, hippocampal neuron L1 insertions were specifically enriched in transcribed neuronal stem cell enhancers and hippocampus genes, increasing their probability of functional relevance. In addition, bias against intronic L1 insertions sense oriented relative to their host gene was observed, perhaps indicating moderate selection against this configuration in vivo. These experiments demonstrate pervasive L1 mosaicism at genomic loci expressed in hippocampal neurons.
Assuntos
Hipocampo/citologia , Elementos Nucleotídeos Longos e Dispersos , Mosaicismo , Neurônios/citologia , Variação Genética , Humanos , Neurogênese , Reação em Cadeia da Polimerase , Bancos de TecidosRESUMO
Implanted biomaterials and devices face compromised functionality and efficacy in the long term owing to foreign body reactions and subsequent formation of fibrous capsules at the implant-tissue interfaces1-4. Here we demonstrate that an adhesive implant-tissue interface can mitigate fibrous capsule formation in diverse animal models, including rats, mice, humanized mice and pigs, by reducing the level of infiltration of inflammatory cells into the adhesive implant-tissue interface compared to the non-adhesive implant-tissue interface. Histological analysis shows that the adhesive implant-tissue interface does not form observable fibrous capsules on diverse organs, including the abdominal wall, colon, stomach, lung and heart, over 12 weeks in vivo. In vitro protein adsorption, multiplex Luminex assays, quantitative PCR, immunofluorescence analysis and RNA sequencing are additionally carried out to validate the hypothesis. We further demonstrate long-term bidirectional electrical communication enabled by implantable electrodes with an adhesive interface over 12 weeks in a rat model in vivo. These findings may offer a promising strategy for long-term anti-fibrotic implant-tissue interfaces.
Assuntos
Materiais Biocompatíveis , Fibrose , Reação a Corpo Estranho , Próteses e Implantes , Adesivos Teciduais , Animais , Feminino , Humanos , Masculino , Camundongos , Ratos , Parede Abdominal , Adsorção , Materiais Biocompatíveis/química , Colo , Eletrodos Implantados , Fibrose/patologia , Fibrose/prevenção & controle , Reação a Corpo Estranho/prevenção & controle , Reação a Corpo Estranho/patologia , Coração , Pulmão , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Ratos Sprague-Dawley , Estômago , Suínos , Fatores de Tempo , Adesivos Teciduais/química , Imunofluorescência , Reprodutibilidade dos Testes , Análise de Sequência de RNARESUMO
Human microbiome research is an actively developing area of inquiry, with ramifications for our lifestyles, our interactions with microbes, and how we treat disease. Advances depend on carefully executed, controlled, and reproducible studies. Here, we provide a Primer for researchers from diverse disciplines interested in conducting microbiome research. We discuss factors to be considered in the design, execution, and data analysis of microbiome studies. These recommendations should help researchers to enter and contribute to this rapidly developing field.
Assuntos
Técnicas Microbiológicas , Microbiota , Animais , Archaea/classificação , Archaea/genética , Archaea/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Guias como Assunto , Humanos , Reação em Cadeia da Polimerase , RibotipagemRESUMO
Models of transcription are often built around a picture of RNA polymerase and transcription factors (TFs) acting on a single copy of a promoter. However, most TFs are shared between multiple genes with varying binding affinities. Beyond that, genes often exist at high copy number-in multiple identical copies on the chromosome or on plasmids or viral vectors with copy numbers in the hundreds. Using a thermodynamic model, we characterize the interplay between TF copy number and the demand for that TF. We demonstrate the parameter-free predictive power of this model as a function of the copy number of the TF and the number and affinities of the available specific binding sites; such predictive control is important for the understanding of transcription and the desire to quantitatively design the output of genetic circuits. Finally, we use these experiments to dynamically measure plasmid copy number through the cell cycle.
Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Modelos Genéticos , Fatores de Transcrição/metabolismo , Escherichia coli/genética , Dosagem de Genes , Regulação Bacteriana da Expressão Gênica , Plasmídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Termodinâmica , Transcrição GênicaRESUMO
Innate lymphoid cells (ILCs) are increasingly appreciated as important participants in homeostasis and inflammation. Substantial plasticity and heterogeneity among ILC populations have been reported. Here we have delineated the heterogeneity of human ILCs through single-cell RNA sequencing of several hundreds of individual tonsil CD127(+) ILCs and natural killer (NK) cells. Unbiased transcriptional clustering revealed four distinct populations, corresponding to ILC1 cells, ILC2 cells, ILC3 cells and NK cells, with their respective transcriptomes recapitulating known as well as unknown transcriptional profiles. The single-cell resolution additionally divulged three transcriptionally and functionally diverse subpopulations of ILC3 cells. Our systematic comparison of single-cell transcriptional variation within and between ILC populations provides new insight into ILC biology during homeostasis, with additional implications for dysregulation of the immune system.
Assuntos
Subunidade alfa de Receptor de Interleucina-7/metabolismo , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/metabolismo , Subpopulações de Linfócitos/metabolismo , Linfócitos/metabolismo , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Imunidade Inata/imunologia , Células Matadoras Naturais/imunologia , Leucócitos Mononucleares/imunologia , Subpopulações de Linfócitos/imunologia , Linfócitos/imunologia , Masculino , Pessoa de Meia-Idade , Tonsila Palatina/citologia , Tonsila Palatina/imunologia , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , Adulto JovemRESUMO
Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8(+) tissue-resident memory T cells (TRM cells) require active transforming growth factor-ß1 (TGF-ß) for epidermal residence. Here we found that integrins αvß6 and αvß8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-ß. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvß6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-ß, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-ß by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Epiderme/imunologia , Mucosa Intestinal/imunologia , Queratinócitos/imunologia , Células de Langerhans/imunologia , Fator de Crescimento Transformador beta/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/citologia , Movimento Celular , Células Epidérmicas , Citometria de Fluxo , Imunofluorescência , Humanos , Imunidade nas Mucosas , Integrinas/imunologia , Mucosa Intestinal/citologia , Intestino Delgado/citologia , Intestino Delgado/imunologia , Células de Langerhans/citologia , Camundongos , Camundongos Knockout , Vison , Reação em Cadeia da Polimerase , Células Estromais , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta1/imunologiaRESUMO
Chain reactions, characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (for example, combustion engines), nuclear and biotechnological (for example, polymerase chain reaction) applications1-5. At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling dominoes and Rube Goldberg machines. On the other hand, the microfluidic lab-on-a-chip (also called a micro-total analysis system)6,7 was visualized as an integrated chip, akin to microelectronic integrated circuits, yet in practice remains dependent on cumbersome peripherals, connections and a computer for automation8-11. Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible12-19. Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are three-dimensionally printed, and powered by the free energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (1) the sequential release of 300 aliquots across chained, interconnected chips, (2) a protocol for severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) antibodies detection in saliva and (3) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ and can form a frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.
Assuntos
COVID-19 , Técnicas Analíticas Microfluídicas , Humanos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Reação em Cadeia da Polimerase , SARS-CoV-2/genéticaRESUMO
Unique molecular identifiers are random oligonucleotide sequences that remove PCR amplification biases. However, the impact that PCR associated sequencing errors have on the accuracy of generating absolute counts of RNA molecules is underappreciated. We show that PCR errors are a source of inaccuracy in both bulk and single-cell sequencing data, and synthesizing unique molecular identifiers using homotrimeric nucleotide blocks provides an error-correcting solution that allows absolute counting of sequenced molecules.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Nucleotídeos , Análise de Sequência de RNA , Oligonucleotídeos/genética , Reação em Cadeia da PolimeraseRESUMO
The molecular mechanisms by which signaling via transforming growth factor-ß (TGF-ß) and interleukin 4 (IL-4) control the differentiation of CD4(+) IL-9-producing helper T cells (TH9 cells) remain incompletely understood. We found here that the DNA-binding inhibitor Id3 regulated TH9 differentiation, as deletion of Id3 increased IL-9 production from CD4(+) T cells. Mechanistically, TGF-ß1 and IL-4 downregulated Id3 expression, and this process required the kinase TAK1. A reduction in Id3 expression enhanced binding of the transcription factors E2A and GATA-3 to the Il9 promoter region, which promoted Il9 transcription. Notably, Id3-mediated control of TH9 differentiation regulated anti-tumor immunity in an experimental melanoma-bearing model in vivo and also in human CD4(+) T cells in vitro. Thus, our study reveals a previously unrecognized TAK1-Id3-E2A-GATA-3 pathway that regulates TH9 differentiation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Proteínas Inibidoras de Diferenciação/imunologia , Interleucina-9/biossíntese , Proteínas de Neoplasias/imunologia , Animais , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Humanos , Proteínas Inibidoras de Diferenciação/genética , Interleucina-9/imunologia , Camundongos , Proteínas de Neoplasias/genética , Reação em Cadeia da Polimerase , Transdução de Sinais/imunologiaRESUMO
Type 2 helper T cells (TH2 cells) produce interleukin 13 (IL-13) when stimulated by papain or house dust mite extract (HDM) and induce eosinophilic inflammation. This innate response is dependent on IL-33 but not T cell antigen receptors (TCRs). While type 2 innate lymphoid cells (ILC2 cells) are the dominant innate producers of IL-13 in naive mice, we found here that helminth-infected mice had more TH2 cells compared to uninfected mice, and thes e cells became major mediators of innate type 2 responses. TH2 cells made important contributions to HDM-induced antigen-nonspecific eosinophilic inflammation and protected mice recovering from infection with Ascaris suum against subsequent infection with the phylogenetically distant nematode Nippostrongylus brasiliensis. Our findings reveal a previously unappreciated role for effector TH2 cells during TCR-independent innate-like immune responses.
Assuntos
Imunidade Inata , Células Th2/imunologia , Animais , Citometria de Fluxo , Helmintíase/imunologia , Helmintos/imunologia , Pulmão/citologia , Pulmão/imunologia , Linfócitos/imunologia , Camundongos , Reação em Cadeia da PolimeraseRESUMO
Treatment with ionizing radiation (IR) can lead to the accumulation of tumor-infiltrating regulatory T cells (Treg cells) and subsequent resistance of tumors to radiotherapy. Here we focused on the contribution of the epidermal mononuclear phagocytes Langerhans cells (LCs) to this phenomenon because of their ability to resist depletion by high-dose IR. We found that LCs resisted apoptosis and rapidly repaired DNA damage after exposure to IR. In particular, we found that the cyclin-dependent kinase inhibitor CDKN1A (p21) was overexpressed in LCs and that Cdkn1a(-/-) LCs underwent apoptosis and accumulated DNA damage following IR treatment. Wild-type LCs upregulated major histocompatibility complex class II molecules, migrated to the draining lymph nodes and induced an increase in Treg cell numbers upon exposure to IR, but Cdkn1a(-/-) LCs did not. Our findings suggest a means for manipulating the resistance of LCs to IR to enhance the response of cutaneous tumors to radiotherapy.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Células de Langerhans/efeitos da radiação , Radiação Ionizante , Linfócitos T Reguladores/efeitos da radiação , Animais , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Citometria de Fluxo , Camundongos , Análise em Microsséries , Reação em Cadeia da Polimerase , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Regulação para CimaRESUMO
The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1ß production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.
Assuntos
Interleucina-4/metabolismo , Macrófagos/metabolismo , Fator de Transcrição STAT6/metabolismo , Animais , Western Blotting , Linhagem Celular , Elementos Facilitadores Genéticos , Citometria de Fluxo , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Citometria de Varredura a Laser , Lipopolissacarídeos/farmacologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Piroptose/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Despite recent advances, many cancers remain refractory to available immunotherapeutic strategies. Emerging evidence indicates that the tolerization of local dendritic cells (DCs) within the tumor microenvironment promotes immune evasion. Here, we have described a mechanism by which melanomas establish a site of immune privilege via a paracrine Wnt5a-ß-catenin-peroxisome proliferator-activated receptor-γ (PPAR-γ) signaling pathway that drives fatty acid oxidation (FAO) in DCs by upregulating the expression of the carnitine palmitoyltransferase-1A (CPT1A) fatty acid transporter. This FAO shift increased the protoporphyrin IX prosthetic group of indoleamine 2,3-dioxgenase-1 (IDO) while suppressing interleukin(IL)-6 and IL-12 cytokine expression, culminating in enhanced IDO activity and the generation of regulatory T cells. We demonstrated that blockade of this pathway augmented anti-melanoma immunity, enhanced the activity of anti-PD-1 antibody immunotherapy, and suppressed disease progression in a transgenic melanoma model. This work implicates a role for tumor-mediated metabolic reprogramming of local DCs in immune evasion and immunotherapy resistance.
Assuntos
Células Dendríticas/metabolismo , Melanoma/imunologia , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo , Animais , Linhagem Celular , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Ácidos Graxos/metabolismo , Feminino , Citometria de Fluxo , Immunoblotting , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Transgênicos , PPAR gama/metabolismo , Comunicação Parácrina/fisiologia , Reação em Cadeia da Polimerase , Transdução de Sinais/fisiologiaRESUMO
Pathogenic simian immunodeficiency virus (SIV) infection is associated with enteropathy, which likely contributes to AIDS progression. To identify candidate etiologies for AIDS enteropathy, we used next-generation sequencing to define the enteric virome during SIV infection in nonhuman primates. Pathogenic, but not nonpathogenic, SIV infection was associated with significant expansion of the enteric virome. We identified at least 32 previously undescribed enteric viruses during pathogenic SIV infection and confirmed their presence by using viral culture and PCR testing. We detected unsuspected mucosal adenovirus infection associated with enteritis as well as parvovirus viremia in animals with advanced AIDS, indicating the pathogenic potential of SIV-associated expansion of the enteric virome. No association between pathogenic SIV infection and the family-level taxonomy of enteric bacteria was detected. Thus, enteric viral infections may contribute to AIDS enteropathy and disease progression. These findings underline the importance of metagenomic analysis of the virome for understanding AIDS pathogenesis.
Assuntos
Caliciviridae/isolamento & purificação , Intestinos/virologia , Parvoviridae/isolamento & purificação , Picornaviridae/isolamento & purificação , Síndrome de Imunodeficiência Adquirida dos Símios/virologia , Vírus da Imunodeficiência Símia/fisiologia , Animais , Caliciviridae/classificação , Caliciviridae/genética , Chlorocebus aethiops , Fezes/microbiologia , Fezes/virologia , Intestinos/microbiologia , Dados de Sequência Molecular , Parvoviridae/classificação , Parvoviridae/genética , Filogenia , Picornaviridae/classificação , Picornaviridae/genética , Reação em Cadeia da Polimerase , Síndrome de Imunodeficiência Adquirida dos Símios/microbiologia , Vírus da Imunodeficiência Símia/patogenicidadeRESUMO
BACKGROUND: Recent developments in CRISPR/Cas9 genome-editing tools have facilitated the introduction of precise alleles, including genetic intervals spanning several kilobases, directly into the embryo. However, the introduction of donor templates, via homology directed repair, can be erroneous or incomplete and these techniques often produce mosaic founder animals. Thus, newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the interval to be sequenced, together with the mosaic nature of founders. METHODOLOGY/PRINCIPAL FINDINGS: In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore Technologies long-read sequencing at the targeted locus and found that the achievable depth of sequencing is sufficient to offset the sequencing error rate associated with the technology used to validate targeted regions of interest. We have assembled an analysis workflow that facilitates interrogating the entire length of a targeted segment in a single read, to confirm that the intended mutant sequence is present in both heterozygous animals and mosaic founders. We used this workflow to compare the output of PCR-based and Cas9 capture-based targeted sequencing for validation of edited alleles. CONCLUSION: Targeted long-read sequencing supports in-depth characterisation of all experimental models that aim to produce knock-in or conditional alleles, including those that contain a mix of genome-edited alleles. PCR- or Cas9 capture-based modalities bring different advantages to the analysis.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Sistemas CRISPR-Cas/genética , Alelos , Edição de Genes/métodos , Reparo de DNA por Recombinação , Reação em Cadeia da PolimeraseRESUMO
Soon after its introduction in 1987, polymerase chain reaction (PCR) has become a technique widely employed in diagnostic medical devices and forensic science with the intention of amplifying genetic information. PCR prescribes that each of its cycles must include a heating subprocess at 95 °C or more (denominated DNA denaturation and provided for allowing a claimed orderly separation of the two complementary nucleotides strands), which can produce significant damage to DNA, caused by high-speed collisions with surrounding molecules. Since such disruption should be prevented in order to reliably employ PCR, a study of the mechanics of such loss of structural integrity is herein presented, preceded by a review of the fundamental literature which has elucidated the effects of molecular agitation on DNA fragmentation. The main conclusion of this retrospective survey is that the body of examined theoretical and experimental evidence consistently and redundantly confirms scarce resilience and significant loss of structural integrity when DNA is heated at temperatures above 90 °C, even for 1 minute. Such conclusion contradicts the claimed paradigm of PCR fidelity and raises the concern that, at least for long sequences, if PCR can amplify some information, such amplified information may be unreliable for diagnostic or forensic applications, since it originates from sequences of nucleotides subjected to random fragmentation and reaggregation. Such a low-reliability scenario should be preventively considered in the various fields where DNA amplification methodologies are employed which provide for high-temperature heating under conditions equal to or similar to those prescribed by the PCR protocols reviewed in this study.
Assuntos
DNA , Reação em Cadeia da Polimerase , DNA/química , Temperatura Alta , Humanos , Reprodutibilidade dos Testes , Calefação , Desnaturação de Ácido NucleicoRESUMO
Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew-$t$ distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app.
Assuntos
Neoplasias , Ácidos Nucleicos , Humanos , Reação em Cadeia da Polimerase/métodos , Benchmarking , Biópsia LíquidaRESUMO
Researchers and clinicians often rely on molecular assays like PCR to identify and monitor viral infections, instead of the resource-prohibitive gold standard of viral culture. However, it remains unclear when (if ever) PCR measurements of viral load are reliable indicators of replicating or infectious virus. The recent popularity of PCR protocols targeting subgenomic RNA for SARS-CoV-2 has caused further confusion, as the relationships between subgenomic RNA and standard total RNA assays are incompletely characterized and opinions differ on which RNA type better predicts culture outcomes. Here, we explore these issues by comparing total RNA, subgenomic RNA, and viral culture results from 24 studies of SARS-CoV-2 in non-human primates (including 2167 samples from 174 individuals) using custom-developed Bayesian statistical models. On out-of-sample data, our best models predict subgenomic RNA positivity from total RNA data with 91% accuracy, and they predict culture positivity with 85% accuracy. Further analyses of individual time series indicate that many apparent prediction errors may arise from issues with assay sensitivity or sample processing, suggesting true accuracy may be higher than these estimates. Total RNA and subgenomic RNA showed equivalent performance as predictors of culture positivity. Multiple cofactors (including exposure conditions, host traits, and assay protocols) influence culture predictions, yielding insights into biological and methodological sources of variation in assay outcomes-and indicating that no single threshold value applies across study designs. We also show that our model can accurately predict when an individual is no longer infectious, illustrating the potential for future models trained on human data to guide clinical decisions on case isolation. Our work shows that meta-analysis of in vivo data can overcome longstanding challenges arising from limited sample sizes and can yield robust insights beyond those attainable from individual studies. Our analytical pipeline offers a framework to develop similar predictive tools in other virus-host systems, including models trained on human data, which could support laboratory analyses, medical decisions, and public health guidelines.