RESUMO
Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.
Assuntos
Haplótipos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias/genética , Análise de Sequência de DNA/métodos , DNA/genética , Genoma Humano , Variação Estrutural do Genoma , Células Germinativas , Humanos , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/genética , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of ~2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more precision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100,000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
Assuntos
DNA/genética , Dosagem de Genes/genética , Sequenciamento de Nucleotídeos em Larga Escala , Reação em Cadeia da Polimerase , HumanosRESUMO
We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instrument's response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.
Assuntos
Bacillus anthracis/isolamento & purificação , Monitoramento Ambiental/métodos , Reação em Cadeia da Polimerase/métodos , Yersinia pestis/isolamento & purificação , Bioterrorismo , Monitoramento Ambiental/instrumentação , Reação em Cadeia da Polimerase/instrumentaçãoRESUMO
We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 x 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 microm, respectively, which provides a relatively large surface area (ca. 3 cm(2)) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.
Assuntos
Poluentes Atmosféricos/análise , Bacillus anthracis/genética , DNA Bacteriano/análise , Monitoramento Ambiental/métodos , Aerossóis , Animais , Desenho de Equipamento , Análise de Injeção de Fluxo , Humanos , Microquímica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodosRESUMO
We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.
Assuntos
Aerossóis/análise , Poluentes Atmosféricos/análise , Bactérias/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Monitoramento Ambiental/instrumentação , Imunoensaio/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Medidas de Segurança , Técnicas Biossensoriais/métodos , Bioterrorismo/prevenção & controle , Monitoramento Ambiental/métodos , Desenho de Equipamento , Imunoensaio/métodos , Reação em Cadeia da Polimerase/métodos , Robótica/instrumentação , Robótica/métodos , Integração de SistemasRESUMO
The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.
Assuntos
Microbiologia do Ar , Bacillus anthracis/isolamento & purificação , Toxinas Botulínicas/análise , Monitoramento Ambiental/instrumentação , Imunoensaio/instrumentação , Reação em Cadeia da Polimerase/instrumentação , Yersinia pestis/isolamento & purificação , Aerossóis , DNA/isolamento & purificação , MicroesferasRESUMO
An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.
Assuntos
Bacillus anthracis/química , Guerra Biológica , Monitoramento Ambiental/métodos , Yersinia pestis/química , Anticorpos Antibacterianos/imunologia , Bacillus anthracis/imunologia , Bacillus anthracis/patogenicidade , Monitoramento Ambiental/instrumentação , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Imunoensaio/instrumentação , Imunoensaio/métodos , Microesferas , Fatores de Tempo , Yersinia pestis/imunologia , Yersinia pestis/patogenicidadeRESUMO
The bidirectional Glenn shunt has been successfully applied as an adjunct to ventricular septal defect closure and pulmonary valvulotomy to treat congenitally corrected transposition of the great arteries (ccTGA). The purpose of this study was to examine the volume and pressure unloading effects of the bidirectional Glenn shunt on the hypertrophied pulmonary ventricle in a canine model of ccTGA. Five beagles underwent survival surgery to band the pulmonary artery. Three months later, a polytetrafluoroethylene graft was anastomosed to the superior vena cava and right pulmonary artery. The graft or superior vena cava was clamped to create the normal or bidirectional Glenn circulation, and hemodynamic data were recorded. The bidirectional Glenn shunt significantly reduced right ventricular volume loading and stroke work. Dogs with normal pre-bidirectional Glenn cardiac outputs had greatly reduced right ventricular volumes and pressures with the bidirectional Glenn shunt. Dogs with pre-bidirectional Glenn right ventricular dysfunction had moderate volume but no pressure decreases with the bidirectional Glenn shunt owing to improved left ventricular output. In these dogs it is likely that the decreased level of pressure and volume unloading is because of a concomitant improvement in left ventricular output post-bidirectional Glenn shunt placement. The bidirectional Glenn shunt is effective at unloading the right ventricle in a canine model of ccTGA.
Assuntos
Derivação Arteriovenosa Cirúrgica/métodos , Procedimentos Cirúrgicos Cardíacos/métodos , Hipertrofia Ventricular Direita/fisiopatologia , Circulação Pulmonar/fisiologia , Transposição dos Grandes Vasos/cirurgia , Animais , Animais Recém-Nascidos , Débito Cardíaco , Modelos Animais de Doenças , Cães , Feminino , Seguimentos , Hemodinâmica/fisiologia , Masculino , Período Pós-Operatório , Probabilidade , Valores de Referência , Medição de Risco , Sensibilidade e Especificidade , Volume Sistólico , Função Ventricular Direita/fisiologiaRESUMO
We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system is designed to provide early warning to civilians in the event of a terrorist attack. The final APDS will be completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. The system performance (current capabilities include aerosol collection, multiplexed immunoassays, sample archiving, data reporting, and alarming) was evaluated in a field test conducted in a Biosafety Level 3 facility, where the system was challenged with, and detected, a series of aerosolized releases containing two live, virulent biological threat agents (Bacillus anthracis and Yersinia pestis). Results presented here represent the first autonomous, simultaneous measurement of these agents.