RESUMEN
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.
Asunto(s)
ADN/química , Microscopía Fluorescente/métodos , Reacción en Cadena de la Polimerasa , Algoritmos , Secuencia de Bases , Línea Celular , Difusión Facilitada/genética , Femenino , Colorantes Fluorescentes/química , Humanos , Nucleótidos/química , Fotones , Coloración y Etiquetado/métodosRESUMEN
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.
Asunto(s)
Bacterias/clasificación , Benchmarking/métodos , Hongos/clasificación , Metagenoma/genética , Metagenómica/métodos , Virus/clasificación , Bacterias/genética , Bases de Datos Genéticas , Hongos/genética , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Programas Informáticos , Virus/genéticaRESUMEN
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.
Asunto(s)
Hipocampo/citología , Elementos de Nucleótido Esparcido Largo , Mosaicismo , Neuronas/citología , Variación Genética , Humanos , Neurogénesis , Reacción en Cadena de la Polimerasa , Bancos de TejidosRESUMEN
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.
Asunto(s)
Materiales Biocompatibles , Fibrosis , Reacción a Cuerpo Extraño , Prótesis e Implantes , Adhesivos Tisulares , Animales , Femenino , Humanos , Masculino , Ratones , Ratas , Pared Abdominal , Adsorción , Materiales Biocompatibles/química , Colon , Electrodos Implantados , Fibrosis/patología , Fibrosis/prevención & control , Reacción a Cuerpo Extraño/prevención & control , Reacción a Cuerpo Extraño/patología , Corazón , Pulmón , Ratones Endogámicos C57BL , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Ratas Sprague-Dawley , Estómago , Porcinos , Factores de Tiempo , Adhesivos Tisulares/química , Técnica del Anticuerpo Fluorescente , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Técnicas Microbiológicas , Microbiota , Animales , Archaea/clasificación , Archaea/genética , Archaea/aislamiento & purificación , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Guías como Asunto , Humanos , Reacción en Cadena de la Polimerasa , RibotipificaciónRESUMEN
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.
Asunto(s)
Escherichia coli/metabolismo , Expresión Génica , Modelos Genéticos , Factores de Transcripción/metabolismo , Escherichia coli/genética , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Termodinámica , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Subunidad alfa del Receptor de Interleucina-7/metabolismo , Células Asesinas Naturales/metabolismo , Leucocitos Mononucleares/metabolismo , Subgrupos Linfocitarios/metabolismo , Linfocitos/metabolismo , Adulto , Anciano , Niño , Preescolar , Femenino , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Inmunidad Innata/inmunología , Células Asesinas Naturales/inmunología , Leucocitos Mononucleares/inmunología , Subgrupos Linfocitarios/inmunología , Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Tonsila Palatina/citología , Tonsila Palatina/inmunología , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ARN , Adulto JovenRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epidermis/inmunología , Mucosa Intestinal/inmunología , Queratinocitos/inmunología , Células de Langerhans/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/citología , Movimiento Celular , Células Epidérmicas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunidad Mucosa , Integrinas/inmunología , Mucosa Intestinal/citología , Intestino Delgado/citología , Intestino Delgado/inmunología , Células de Langerhans/citología , Ratones , Ratones Noqueados , Visón , Reacción en Cadena de la Polimerasa , Células del Estroma , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
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.
Asunto(s)
COVID-19 , Técnicas Analíticas Microfluídicas , Humanos , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Reacción en Cadena de la Polimerasa , SARS-CoV-2/genéticaRESUMEN
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.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Nucleótidos , Análisis de Secuencia de ARN , Oligonucleótidos/genética , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Proteínas Inhibidoras de la Diferenciación/inmunología , Interleucina-9/biosíntesis , Proteínas de Neoplasias/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Citometría de Flujo , Humanos , Proteínas Inhibidoras de la Diferenciación/genética , Interleucina-9/inmunología , Ratones , Proteínas de Neoplasias/genética , Reacción en Cadena de la Polimerasa , Transducción de Señal/inmunologíaRESUMEN
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.
Asunto(s)
Inmunidad Innata , Células Th2/inmunología , Animales , Citometría de Flujo , Helmintiasis/inmunología , Helmintos/inmunología , Pulmón/citología , Pulmón/inmunología , Linfocitos/inmunología , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células de Langerhans/efectos de la radiación , Radiación Ionizante , Linfocitos T Reguladores/efectos de la radiación , Animales , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Citometría de Flujo , Ratones , Análisis por Micromatrices , Reacción en Cadena de la Polimerasa , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Regulación hacia ArribaRESUMEN
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.
Asunto(s)
Interleucina-4/metabolismo , Macrófagos/metabolismo , Factor de Transcripción STAT6/metabolismo , Animales , Western Blotting , Línea Celular , Elementos de Facilitación Genéticos , Citometría de Flujo , Regulación de la Expresión Génica , Inflamasomas/metabolismo , Citometría de Barrido por Láser , Lipopolisacáridos/farmacología , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa , Piroptosis/genética , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Células Dendríticas/metabolismo , Melanoma/inmunología , Proteína Wnt-5a/metabolismo , beta Catenina/metabolismo , Animales , Línea Celular , Células Dendríticas/inmunología , Ensayo de Inmunoadsorción Enzimática , Ácidos Grasos/metabolismo , Femenino , Citometría de Flujo , Immunoblotting , Masculino , Melanoma/metabolismo , Ratones , Ratones Transgénicos , PPAR gamma/metabolismo , Comunicación Paracrina/fisiología , Reacción en Cadena de la Polimerasa , Transducción de Señal/fisiologíaRESUMEN
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.
Asunto(s)
Caliciviridae/aislamiento & purificación , Intestinos/virología , Parvoviridae/aislamiento & purificación , Picornaviridae/aislamiento & purificación , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/fisiología , Animales , Caliciviridae/clasificación , Caliciviridae/genética , Chlorocebus aethiops , Heces/microbiología , Heces/virología , Intestinos/microbiología , Datos de Secuencia Molecular , Parvoviridae/clasificación , Parvoviridae/genética , Filogenia , Picornaviridae/clasificación , Picornaviridae/genética , Reacción en Cadena de la Polimerasa , Síndrome de Inmunodeficiencia Adquirida del Simio/microbiología , Virus de la Inmunodeficiencia de los Simios/patogenicidadRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Animales , Sistemas CRISPR-Cas/genética , Alelos , Edición Génica/métodos , Reparación del ADN por Recombinación , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
ADN , Reacción en Cadena de la Polimerasa , ADN/química , Calor , Humanos , Reproducibilidad de los Resultados , Calefacción , Desnaturalización de Ácido NucleicoRESUMEN
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.
Asunto(s)
Neoplasias , Ácidos Nucleicos , Humanos , Reacción en Cadena de la Polimerasa/métodos , Benchmarking , Biopsia LíquidaRESUMEN
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.