RESUMEN
BACKGROUND: Next Generation Sequencing has proven to be an exceptionally powerful tool in the field of genomics and transcriptomics. With recent development it is nowadays possible to analyze ultra-low input sample material down to single cells. Nevertheless, investigating such sample material often limits the analysis to either the genome or transcriptome. We describe here a combined analysis of both types of nucleic acids from the same sample material. METHODS: The method described enables the combined preparation of amplified cDNA as well as amplified whole-genome DNA from an ultra-low input sample material derived from a sub-colony of in-vitro cultivated human embryonic stem cells. cDNA is prepared by the application of oligo-dT coupled magnetic beads for mRNA capture, first strand synthesis and 3'-tailing followed by PCR. Whole-genome amplified DNA is prepared by Phi29 mediated amplification. Illumina sequencing is applied to short fragment libraries prepared from the amplified samples. RESULTS: We developed a protocol which enables the combined analysis of the genome as well as the transcriptome by Next Generation Sequencing from ultra-low input samples. The protocol was evaluated by sequencing sub-colony structures from human embryonic stem cells containing 150 to 200 cells. The method can be adapted to any available sequencing system. CONCLUSIONS: To our knowledge, this is the first report where sub-colonies of human embryonic stem cells have been analyzed both at the genomic as well as transcriptome level. The method of this proof of concept study may find useful practical applications for cases where only a limited number of cells are available, e.g. for tissues samples from biopsies, tumor spheres, circulating tumor cells and cells from early embryonic development. The results we present demonstrate that a combined analysis of genomic DNA and messenger RNA from ultra-low input samples is feasible and can readily be applied to other cellular systems with limited material available.
Asunto(s)
Genoma Humano , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/metabolismo , ARN Mensajero/genética , Biomarcadores , Análisis por Conglomerados , Perfilación de la Expresión Génica , Genómica/métodos , Células Madre Embrionarias Humanas/citología , Humanos , ARN Mensajero/metabolismo , TranscriptomaRESUMEN
In the framework of the EMPIR project traceRadon, stable atmospheres with low-level radon activity concentrations have to be produced for calibrating radon detectors designed to measure outdoor air activity concentrations. The traceable calibration of these detectors at very low activity concentrations is of special interest to the radiation protection, climate observation, and atmospheric research communities. Radiation protection networks (such as the EUropean Radiological Data Exchange Platform (EURDEP)) and atmospheric monitoring networks (such as the Integrated Carbon Observation System (ICOS)) need reliable and accurate radon activity concentration measurements for a variety of reasons, including: the identification of Radon Priority Areas (RPA); improving the sensitivity and reliability of radiological emergency early warning systems (Melintescu et al., 2018); for more reliable application of the Radon Tracer Method (RTM) to estimate greenhouse gas (GHG) emissions; for improved global "baseline" monitoring of changing GHG concentrations and quantification of regional pollution transport (Chambers et al., 2016), (Chambers et al., 2018); and for evaluating mixing and transport parameterisations in regional or global chemical transport models (CTMs) (Zhang et al., 2021), (Chambers et al., 2019). To achieve this goal, low activity sources of radium with a variety of characteristics were produced using different methods. Sources ranging from MBq 226Ra down to several Bq 226Ra were developed and characterised during the evolution of production methods, and uncertainties below 2 % (k=1) were achieved through dedicated detection techniques, even for the lowest activity sources. The uncertainty of the lowest activity sources was improved using a new online measurement technique for which the source and detector were combined in the same device. This Integrated Radon Source Detector device, henceforth an IRSD, reaches a counting efficiency approaching 50 % through detection under quasi 2π sr solid-angle. At the time of this study the IRSD was already produced with 226Ra activities between 2 Bq and 440 Bq. To compare the working performance of the developed sources (i.e., to establish a reference atmosphere), study the stability of the sources, and to establish traceability to national standards, an intercomparison exercise was carried out at the PTB facility. Here we present the various source production techniques, the determination of their radium activity, and determination of their radon emanation (including assigned uncertainties). This includes details of the implementation of the intercomparison set-up, and a discussion of the results of the source characterisations.
RESUMEN
In this work, a novel approach for the standardization of low-level 222Rn emanation is presented. The technique is based on the integration of a 222Rn source, directly, with an α-particle detector, which allows the residual 222Rn to be continuously monitored. Preparation of the device entails thermal physical vapor deposition of 226RaCl2 directly onto the surface of a commercially available ion implanted Si-diode detector, resulting in a thin-layer geometry. This enables continuous collection of well resolved α-particle spectra of the nuclei, decaying within the deposited layer, with a detection efficiency of approximately 0.5 in a quasi 2π geometry. The continuously sampled α-particle spectra are used to derive the emanation by statistical inversion. It is possible to achieve this with high temporal resolution due to the small background and the high counting efficiency of the presented technique. The emanation derived in this way exhibits a dependence on the relative humidity of up to 15% in the range from 20% rH to 90% rH. Traceability to the SI is provided by employing defined solid-angle α-particle spectrometry to characterize the counting efficiency of the modified detectors. The presented technique is demonstrated to apply to a range covering the release of at least 1 to 210 222Rn atoms per second, and it results in SI-traceable emanation values with a combined standard uncertainty not exceeding 2%. This provides a pathway for the realization of reference atmospheres covering typical environmental 222Rn levels and thus drastically improves the realization and the dissemination of the derived unit of the activity concentration concerning 222Rn in air.
Asunto(s)
Contaminantes Radiactivos del Aire , Monitoreo de Radiación , Radón , Contaminantes Radiactivos del Aire/análisis , Radón/análisisRESUMEN
Laser resonance ionization at the RISIKO 30 kV mass separator has been used to produce isotopically and isobarically pure and well quantified 222Rn emanation standards. Based upon laser-spectroscopic preparation studies, ion implantation into aluminum and tungsten targets has been carried out, providing overall implantation efficiencies of 40% up to 60%. The absolute implanted activity of 226Ra was determined by the technique of defined solid-angle α-particle spectrometry, where excellent energy resolution was observed. The 222Rn emanation coefficient of the produced targets was studied using α-particle and γ-ray spectrometry, and yielded results between 0.23 and 0.34, with relative uncertainty on the order of 1%. No dependence exceeding a 1% change of the emanation on humidity could be identified in the range of 15 %rH to 75 %rH, whereas there were hints of a slight correlation between the emanation and temperature. Additionally, and as expected, the emanation coefficient was found to be dependent on the target material as well as the implanted dose.
RESUMEN
New emanation sources for Rn-222 have been developed by electrodeposition of Ra-226 onto stainless-steel discs. With a high resolution of up to 20â¯keV FWHM in the Ra-226 peak at 4.87â¯MeV, defined solid-angle alpha-particle spectrometry is the method of choice to determine the deposited Ra-226 activity. The amount of emanating Rn-222 is determined by gamma-ray spectrometry using HPGe-detectors. The measurement is based on the distorted equilibrium of the Ra-226 decay chain due to Rn-222 emanation. Comparative gamma-ray spectrometric measurements with sealed, Rn-222 tight sources of the same type and geometry make the knowledge of emission probabilities and detection efficiency unnecessary. The new emanation sources allow the production of stable reference atmospheres in the regime below 300 Bqâ m-3 with uncertainties not exceeding 2% for kâ¯=â¯1.
RESUMEN
Xenoturbella and the acoelomorph worms (Xenacoelomorpha) are simple marine animals with controversial affinities. They have been placed as the sister group of all other bilaterian animals (Nephrozoa hypothesis), implying their simplicity is an ancient characteristic [1, 2]; alternatively, they have been linked to the complex Ambulacraria (echinoderms and hemichordates) in a clade called the Xenambulacraria [3-5], suggesting their simplicity evolved by reduction from a complex ancestor. The difficulty resolving this problem implies the phylogenetic signal supporting the correct solution is weak and affected by inadequate modeling, creating a misleading non-phylogenetic signal. The idea that the Nephrozoa hypothesis might be an artifact is prompted by the faster molecular evolutionary rate observed within the Acoelomorpha. Unequal rates of evolution are known to result in the systematic artifact of long branch attraction, which would be predicted to result in an attraction between long-branch acoelomorphs and the outgroup, pulling them toward the root [6]. Other biases inadequately accommodated by the models used can also have strong effects, exacerbated in the context of short internal branches and long terminal branches [7]. We have assembled a large and informative dataset to address this problem. Analyses designed to reduce or to emphasize misleading signals show the Nephrozoa hypothesis is supported under conditions expected to exacerbate errors, and the Xenambulacraria hypothesis is preferred in conditions designed to reduce errors. Our reanalyses of two other recently published datasets [1, 2] produce the same result. We conclude that the Xenacoelomorpha are simplified relatives of the Ambulacraria.
Asunto(s)
Evolución Biológica , Invertebrados/clasificación , Filogenia , Animales , Cordados/clasificación , Equinodermos/clasificación , Invertebrados/anatomía & histologíaRESUMEN
Dioxins and dioxin-like compounds can be analyzed by bioanalytical screening methods to evaluate their biotoxicity. In vitro bioassays, based on 7-ethoxyresorufin-O-deethylase (EROD) and the activity of cytochrome P450 1A1 and the aryl hydrogen receptor (AhR) pathway, are employed for the evaluation of bioanalytical equivalents (BEQ) of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) from a wide variety of sample matrices. Here, we present the evaluation of 11 humic soil samples derived from forest stands across Germany and a comparison of the BEQ values against toxic equivalents (TEQ, PCDD/Fs+PCBs) derived by chemical analysis. BEQ values ranged from 8.8 to 34.1 while TEQ values from 13.9 to 60.5 pg/g dry weight. Additional two subsequent mineral layers were analyzed to identify the BEQ/TEQ gradient vertically, showing a TEQ decrease of 85.1 and 93.8 % from the humic to the first and second mineral layers, respectively. For BEQ values, a decrease as well as an increase was detected. BEQ measurements were performed with and without sample clean-up. Omitting clean-up revealed about 20 times increased BEQ values presumably due to non-persistent bioactive compounds not detected by chemical analysis. The results we present suggest that the EROD assay can be used for the screening of large sample quantities for the identification of samples showing dioxin and dioxin-like contaminations even at low levels, which can then be further analyzed by chemical analysis to identify the congener composition. The study also shows that EROD results give a qualitative image of the contamination. EROD seems to be interfered with cross-contaminants specifically for soils with high biological activity as forest layers.
Asunto(s)
Dioxinas/análisis , Contaminantes del Suelo/análisis , Animales , Línea Celular Tumoral , Citocromo P-450 CYP1A1/química , Bosques , Cromatografía de Gases y Espectrometría de Masas , Alemania , RatasRESUMEN
Recent European regulations have indicated the need for new bioanalytical screening methods capable of monitoring dioxin and dioxin-like compounds in foodstuffs and environmental samples, cost-effectively and with a quicker turnaround. Cryo-cells of the hepatic H4IIE line preserved in 96-well plates were exposed to sample extracts prepared from various foodstuffs and analysed for their content of dioxins and dioxin-like compounds by means of the 7-Ethoxyresorufin-O-Deethylase (EROD)-assay in two laboratories. Assay data were compared between both laboratories and results from instrumental analysis used as a confirmatory method. Additionally, cut-off values for the different studied matrices were derived. The current European regulation regarding methods of analysis for the control of foodstuffs was applied with the aim of determining the feasibility of the cryo-methodology. Results obtained in both laboratories were in congruence with the required validation parameters of the Commission Regulation (EU) No 2017/644. Cut-off values should be established matrix-dependent to reduce the rate of false compliant results and to keep the rate of false non-compliant results under control. In summary, the ready-to-use cryo-assay method for the bioanalytical screening of foodstuffs in control laboratories without cell-culture facilities has successfully proven to be accurate, far quicker and more cost effective than current methods.
Asunto(s)
Técnicas de Química Analítica/métodos , Citocromo P-450 CYP1A1/metabolismo , Dioxinas/análisis , Contaminación de Alimentos/análisis , Congelación , Animales , Línea Celular , Línea Celular Tumoral , Dioxinas/normas , Europa (Continente) , Adhesión a Directriz , Límite de Detección , RatasRESUMEN
Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells) derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A) mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO) database under accession number GSE69471.
RESUMEN
The occurrence of bromocarbazoles and chlorocarbazoles was studied in 86 forest soil samples from different regions in Germany. Carbazole, 3-chlorocarbazole, 3-bromocarbazole and 3,6-dibromocarbazole were qualitatively detected in the humic layer of 59 soil samples with bromocarbazoles reported here for the first time in soil. Furthermore, the halogenated carbazoles, PCDD/Fs and PCBs were detected in the humic and mineral soil horizons (0-5 cm and 5-10 cm) of a subset of 11 soil samples subjected to quantitative analysis. Concentrations ranged from 0.6 to 267.6 ng/g (carbazole); 0.2-7.2 ng/g (3-bromocarbazole); 0.0-9.1 ng/g (3-chlorocarbazole); 0.2-19.8 ng/g (3,6-dibromocarbazole); 0.4-67.6 ng/g (3,6-dichlorocarbazole); 0.0-0.7 ng/g (PCDDs); 0.0-0.3 ng/g (PCDFs) and 0.0-33.7 ng/g (PCBs). Concentrations decreased with depth and correlated positively to total organic carbon (TOC). When it was based on TOC%, an increase in concentration with depth was observed in most soil samples. With respect to dioxin-like toxicity, 3-bromocarbazole, 3-chlorocarbazole, 3,6-dibromocarbazole and 3,6-dichlorocarbazoles caused induction of CYP1A1-dependent EROD activity in HII4E rat hepatoma cell line. Their relative effect potency after 72 h exposure ranged from 0.00005 to 0.00013 and was directly related to the degree of halogenation with 2,3,7,8-tetrachlorodibenzo-p-dioxin as reference. Furthermore, their contribution to overall soil dioxin-like toxicity was not significant in comparison to PCDD/Fs and PCBs though the sum toxic equivalency was limited to three halogenated carbazole congeners. Bromocarbazoles and chlorocarbazoles are emerging dioxin-like toxic environmental contaminants with potential for wide distribution occurring simultaneously with PCDD/Fs and PCBs.
Asunto(s)
Carbazoles/análisis , Dioxinas/análisis , Monitoreo del Ambiente/métodos , Contaminantes del Suelo/análisis , Animales , Benzofuranos/análisis , Carbazoles/química , Citocromo P-450 CYP1A1/metabolismo , Dioxinas/química , Contaminación Ambiental/análisis , Bosques , Alemania , Halogenación , Bifenilos Policlorados/análisis , Dibenzodioxinas Policloradas , Ratas , Suelo/químicaRESUMEN
The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 41/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
Asunto(s)
Biotecnología/métodos , ADN/análisis , ADN/genética , Animales , Química Clic , Exoma/genética , Humanos , Espectrometría de Masas , Análisis de Secuencia de ADNRESUMEN
Since the introduction of next-generation sequencing, several techniques have been developed to selectively enrich and sequence specific parts of the genome at high coverage. These techniques include enzymatic methods employing molecular inversion probes, PCR based approaches, hybrid capture, and in-solution capture. In-solution capture employs RNA probes transcribed from a pool of DNA template oligos designed to match regions of interest to specifically bind and enrich genomic DNA fragments. This method is highly efficient, especially if genomic target regions are large in size or quantity. Diverse in-solution capture kits are available, but are costly when large sample numbers need to be analyzed. Here we present a cost-effective strategy for the design of custom DNA libraries, their transcription into RNA libraries, and application for in-solution capture. We show the efficacy by comparing the method to a commercial kit and further demonstrate that emulsion PCR can be used for bias free amplification and virtual immortalization of DNA template libraries.
Asunto(s)
Biología Computacional/métodos , Biblioteca de Genes , Análisis de Secuencia de ADN/métodos , Mapeo Cromosómico/métodos , Bases de Datos Genéticas , Genes Relacionados con las Neoplasias , Sitios Genéticos , Humanos , Oligonucleótidos/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido SimpleRESUMEN
In this review, we discuss the latest targeted enrichment methods and aspects of their utilization along with second-generation sequencing for complex genome analysis. In doing so, we provide an overview of issues involved in detecting genetic variation, for which targeted enrichment has become a powerful tool. We explain how targeted enrichment for next-generation sequencing has made great progress in terms of methodology, ease of use and applicability, but emphasize the remaining challenges such as the lack of even coverage across targeted regions. Costs are also considered versus the alternative of whole-genome sequencing which is becoming ever more affordable. We conclude that targeted enrichment is likely to be the most economical option for many years to come in a range of settings.
Asunto(s)
Genoma , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Animales , Secuencia de Bases , HumanosRESUMEN
Massively parallel sequencing (MPS) is rapidly evolving and is starting to be utilized by the clinical field as well as diagnostics. We describe major recent advances that have come about as a result of the application of MPS in the biomedical field and the first approaches in medical genetics that have made use of MPS. Without any doubt, MPS has proven to be a very powerful technique. To unravel the capabilities of MPS for patient care, the most important aspect for the acceptance of MPS within clinics and diagnostics is to guarantee that the large amount of data undergoes vitally important analyses and interpretation and is securely managed.
RESUMEN
Salmonella enterica subsp. enterica serovar identification is of great importance with respect to outbreak monitoring and case verification. Therefore rapid, sensitive and cost efficient detection of Salmonella spp. is indispensable within microbiology labs. To amalgamate single tube isolate identification with Salmonella typing, we developed the high-throughput Universal Probe Salmonella Serotyping (UPSS) technique based on nano liter PCR. In comparison to the classical approach, where O- and H-antisera are applied, the UPSS relies on specific gene content amplification of Salmonella spp. by a universal TaqMan assay for all markers and identification of the specific amplicon pattern. To enable high-throughput technology we employed a chip format containing 1024 wells loaded by an automated liquid-handling system which allowed us to perform TaqMan PCR reactions in volumes of 100nL per well. Herein we present proof of principle of the UPSS method by the use of a test panel of 100 previously serotyped Salmonella isolates to successfully verify the usability, accuracy and feasibility of the newly developed UPSS approach. We found that the methodology of the UPSS technology is capable of unequivocally identifying 30 Salmonella serotypes on a single chip within 3 hours but can be highly parallelized by the use of multiple PCR machines. Therefore the UPSS method offers a robust and straightforward molecular alternative for Salmonella detection and typing that saves expensive chemistry and can be easily automated.
Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Reacción en Cadena de la Polimerasa/métodos , Salmonella enterica/clasificación , Salmonella enterica/genética , Serotipificación/métodos , Animales , Genotipo , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Epidemiología Molecular/métodos , Sondas de Oligonucleótidos/genética , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiología , Factores de TiempoRESUMEN
Ojoplano (Opo) is a morphogenetic gene playing an important role during embryogenesis in medaka. This report focuses on the identification and characterization of the mouse Opo gene. We examined Opo expression by whole-mount in situ hybridization and in situ hybridization on sagittal sections during mouse embryogenesis. First expression in whole-mounts was detected at Theiler stages 15-17 (E 9.5-10.5dpc) as a spotted specific staining in migrating neural crest cells and in placodal structures. A complex expression pattern was observed in Theiler stage 22-23 (E 14.5dpc) in sagittal sections, including expression in skeletal structures (skull, vertebrae, ribs, bones of the locomotor system), in the nasal region, the heart and the eye. Fusion proteins revealed the localization of OPO within the cytoplasm with a reticular distribution that largely overlapped with the endoplasmic reticulum. Opo shows homology to human transcripts linked to a hereditary craniofacial malformation, orofacial cleft 1 (OFC1). The expression of mouse Opo in neural crest derivatives and skull elements further supports this link.
Asunto(s)
Embrión de Mamíferos/embriología , Proteínas del Ojo/genética , Morfogénesis/genética , Cresta Neural/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Desarrollo Embrionario/genética , Proteínas del Ojo/fisiología , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Oryzias/genética , Proteínas/fisiología , Alineación de SecuenciaRESUMEN
Large numbers of protein expression changes are usually observed in mouse models for neurodegenerative diseases, even when only a single gene was mutated in each case. To study the effect of gene dose alterations on the cellular proteome, we carried out a proteomic investigation on murine embryonic stem cells that either overexpressed individual genes or displayed aneuploidy over a genomic region encompassing 14 genes. The number of variant proteins detected per cell line ranged between 70 and 110, and did not correlate with the number of modified genes. In cell lines with single gene mutations, up and down-regulated proteins were always in balance in comparison to parental cell lines regarding number as well as concentration of differentially expressed proteins. In contrast, dose alteration of 14 genes resulted in an unequal number of up and down-regulated proteins, though the balance was kept at the level of protein concentration. We propose that the observed protein changes might partially be explained by a proteomic network response. Hence, we hypothesize the existence of a class of "balancer" proteins within the proteomic network, defined as proteins that buffer or cushion a system, and thus oppose multiple system disturbances. Through database queries and resilience analysis of the protein interaction network, we found that potential balancer proteins are of high cellular abundance, possess a low number of direct interaction partners, and show great allelic variation. Moreover, balancer proteins contribute more heavily to the network entropy, and thus are of high importance in terms of system resilience. We propose that the "elasticity" of the proteomic regulatory network mediated by balancer proteins may compensate for changes that occur under diseased conditions.