High-Throughput SNP Genotyping by SBE/SBH

Despite much progress over the past decade, current Single Nucleotide Polymorphism (SNP) genotyping technologies still offer an insufficient degree of multiplexing when required to handle user-selected sets of SNPs. In this paper we propose a new genotyping assay architecture combining multiplexed solution-phase single-base extension (SBE) reactions with sequencing by hybridization (SBH) using universal DNA arrays such as all $k$-mer arrays. In addition to PCR amplification of genomic DNA, SNP genotyping using SBE/SBH assays involves the following steps: (1) Synthesizing primers complementing the genomic sequence immediately preceding SNPs of interest; (2) Hybridizing these primers with the genomic DNA; (3) Extending each primer by a single base using polymerase enzyme and dideoxynucleotides labeled with 4 different fluorescent dyes; and finally (4) Hybridizing extended primers to a universal DNA array and determining the identity of the bases that extend each primer by hybridization pattern analysis. Our contributions include a study of multiplexing algorithms for SBE/SBH genotyping assays and preliminary experimental results showing the achievable tradeoffs between the number of array probes and primer length on one hand and the number of SNPs that can be assayed simultaneously on the other. Simulation results on datasets both randomly generated and extracted from the NCBI dbSNP database suggest that the SBE/SBH architecture provides a flexible and cost-effective alternative to genotyping assays currently used in the industry, enabling genotyping of up to hundreds of thousands of user-specified SNPs per assay.Comment: 19 page

CAD Tools for DNA Micro-Array Design, Manufacture and Application

Motivation: As the human genome project progresses and some microbial and eukaryotic genomes are recognized, numerous biotechnological processes have attracted increasing number of biologists, bioengineers and computer scientists recently. Biotechnological processes profoundly involve production and analysis of highthroughput experimental data. Numerous sequence libraries of DNA and protein structures of a large number of micro-organisms and a variety of other databases related to biology and chemistry are available. For example, microarray technology, a novel biotechnology, promises to monitor the whole genome at once, so that researchers can study the whole genome on the global level and have a better picture of the expressions among millions of genes simultaneously. Today, it is widely used in many fields- disease diagnosis, gene classification, gene regulatory network, and drug discovery. For example, designing organism specific microarray and analysis of experimental data require combining heterogeneous computational tools that usually differ in the data format; such as, GeneMark for ORF extraction, Promide for DNA probe selection, Chip for probe placement on microarray chip, BLAST to compare sequences, MEGA for phylogenetic analysis, and ClustalX for multiple alignments. Solution: Surprisingly enough, despite huge research efforts invested in DNA array applications, very few works are devoted to computer-aided optimization of DNA array design and manufacturing. Current design practices are dominated by ad-hoc heuristics incorporated in proprietary tools with unknown suboptimality. This will soon become a bottleneck for the new generation of high-density arrays, such as the ones currently being designed at Perlegen [109]. The goal of the already accomplished research was to develop highly scalable tools, with predictable runtime and quality, for cost-effective, computer-aided design and manufacturing of DNA probe arrays. We illustrate the utility of our approach by taking a concrete example of combining the design tools of microarray technology for Harpes B virus DNA data

CRMAGE: CRISPR Optimized MAGE Recombineering

A bottleneck in metabolic engineering and systems biology approaches is the lack of efficient genome engineering technologies. Here, we combine CRISPR/Cas9 and λ Red recombineering based MAGE technology (CRMAGE) to create a highly efficient and fast method for genome engineering of Escherichia coli. Using CRMAGE, the recombineering efficiency was between 96.5% and 99.7% for gene recoding of three genomic targets, compared to between 0.68% and 5.4% using traditional recombineering. For modulation of protein synthesis (small insertion/RBS substitution) the efficiency was increased from 6% to 70%. CRMAGE can be multiplexed and enables introduction of at least two mutations in a single round of recombineering with similar efficiencies. PAM-independent loci were targeted using degenerate codons, thereby making it possible to modify any site in the genome. CRMAGE is based on two plasmids that are assembled by a USER-cloning approach enabling quick and cost efficient gRNA replacement. CRMAGE furthermore utilizes CRISPR/Cas9 for efficient plasmid curing, thereby enabling multiple engineering rounds per day. To facilitate the design process, a web-based tool was developed to predict both the λ Red oligos and the gRNAs. The CRMAGE platform enables highly efficient and fast genome editing and may open up promising prospective for automation of genome-scale engineering

From Molecular Blocks To Bioanalytical Assays: Combining Lanthanide Luminescence and Bioaffinity Binders for Protein Detection

Measuring protein biomarkers from sample matrix, such as plasma, is one of the basic tasks in clinical diagnostics. Bioanalytical assays used for the measuring should be able to measure proteins with high sensitivity and specificity. Furthermore, multiplexing capability would also be advantageous. To ensure the utility of the diagnostic test in point-of-care setting, additional requirements such as short turn-around times, ease-ofuse and low costs need to be met. On the other hand, enhancement of assay sensitivity could enable exploiting novel biomarkers, which are present in very low concentrations and which the current immunoassays are unable to measure. Furthermore, highly sensitive assays could enable the use of minimally invasive sampling. In the development of high-sensitivity assays the label technology and affinity binders are in pivotal role. Additionally, innovative assay designs contribute to the obtained sensitivity and other characteristics of the assay as well as its applicability. The aim of this thesis was to study the impact of assay components on the performance of both homogeneous and heterogeneous assays. Applicability of two different lanthanide-based label technologies, upconverting nanoparticles and switchable lanthanide luminescence, to protein detection was explored. Moreover, the potential of recombinant antibodies and aptamers as alternative affinity binders were evaluated. Additionally, alternative conjugation chemistries for production of the labeled binders were studied. Different assay concepts were also evaluated with respect to their applicability to point-of-care testing, which requires simple yet sensitive methods. The applicability of upconverting nanoparticles to the simultaneous quantitative measurement of multiple analytes using imaging-based detection was demonstrated. Additionally, the required instrumentation was relatively simple and inexpensive compared to other luminescent lanthanide-based labels requiring time-resolved measurement. The developed homogeneous assays exploiting switchable lanthanide luminescence were rapid and simple to perform and thus applicable even to point-ofcare testing. The sensitivities of the homogeneous assays were in the picomolar range, which are still inadequate for some analytes, such as cardiac troponins, requiring ultralow limits of detection. For most analytes, however, the obtained limits of detection were sufficient. The use of recombinant antibody fragments and aptamers as binders allowed site-specific and controlled covalent conjugation to construct labeled binders reproducibly either by using chemical modification or recombinant technology. Luminescent lanthanide labels were shown to be widely applicable for protein detection in various assay setups and to contribute assay sensitivity.Proteiinien mittaaminen biologisesta näytteestä, kuten plasmasta, on kliinisen diagnostiikan perustehtäviä. Kliinisessä diagnostiikassa käytettävien bioanalyyttisten määritysten tulisi olla herkkiä ja tarkkoja, jotta ne voivat mitata proteiineja näytteestä luotettavasti. Lisäksi olisi hyödyllistä pystyä mittaamaan samasta näytteestä monta analyyttiä yhtäaikaisesti. Vieritestaukseen tarkoitettujen testien tulisi edellä mainittujen ominaisuuksien ohella olla nopeita, helppokäyttöisiä ja edullisia. Toisaalta nykyistä merkittävästi herkempien määritysten kehittäminen mahdollistaisi uusien, hyvin pieninä pitoisuuksina esiintyvien biomerkkiaineiden käytön diagnostiikassa. Erittäin herkät määritykset voisivat myös mahdollistaa helposti saatavilla olevien näytemateriaalien käytön. Määrityksen herkkyyteen ja muihin ominaisuuksiin vaikuttavia tekijöitä ovat käytetty leimateknologia sekä sitojamolekyylit. Lisäksi määrityskonseptin valinnalla voidaan vaikuttaa testin suorituskykyyn ja sen soveltuvuuteen esimerkiksi vieritestausolosuhteisiin. Tässä väitöskirjatyössä tutkittiin kahden luminoivan lantanidileimateknologian soveltuvuutta proteiinien mittaamiseen. Yhdessä osatyössä tutkittiin upkonvertoivien eli käänteisviritteisten luminoivien nanopartikkelien käyttökelpoisuutta monianalyyttimäärityksiin ja kolmessa osatyössä kehitettiin nopeita erotusvapaita määrityksiä mallianalyyteille hyödyntäen kytkeytyvää lantanidiluminesenssia. Osatöissä selvitettiin myös rekombinanttisten vasta-ainefragmenttien ja aptameerien soveltuvuutta proteiinipitoisuuksien mittaamiseen. tutkittiin erilaisia konjugointitapoja leiman ja sitojamolekyylin yhdistämiseksi. Lisäksi arvioitiin eri määritystapojen soveltuvuutta vieritestaussovelluksiin. Väitöskirjatyössä osoitettiin, että käänteisviritteisiä nanopartikkeleita voidaan hyödyntää usean analyytin yhtäaikaiseen mittaamiseen käyttämällä kuvantavaa mittaustapaa. Tällöin voidaan käyttää yksinkertaisempaa laitteistoa, sillä toisin kuin muut luminoivat lantanidileimat, käänteisviritteiset nanopartikkelit eivät edellytä aikaerotteista mittaustapaa. Kehitetyt erotusvapaat kytkeytyvää lantanidiluminesenssia hyödyntävät määritykset olivat nopeita ja yksinkertaisia suorittaa, ja siten soveltuvia jopa vieritesteihin. Erotusvapaiden määritysten havaintorajat olivat pikomolaarisella alueella, mikä ei ole riittävän herkkä kaikille analyyteille kuten esimerkiksi sydänspesifisille troponiineille, joiden mittaamiseen vaaditaan erittäin herkkiä testejä. Vasta-ainefragmenttien ja oligonukleotidipohjaisten aptameerien käyttö mahdollisti paikkaspesifisen ja kontrolloidun leima-sitojamolekyyli-konjugaatin muodostamisen joko kemiallisesti tai rekombinanttiteknologiaa käyttäen. Käänteisviritteisten luminoivien nanopartikkelien ja kytkeytyvien lantanidileimojen osoitettiin soveltuvan proteiinien detektioon monenlaisissa määrityskonsepteissa.Siirretty Doriast

Study of Strategies for Genetic Variant Discrimination and Detection by Optosensing

Tesis por compendio[ES] La medicina actual se dirige hacia un enfoque más personalizado basándose en el diagnóstico molecular del paciente a través del estudio de biomarcadores específicos. Aplicando este principio molecular, el diagnóstico, pronóstico y selección de la terapia se apoyan en la identificación de variaciones específicas del genoma humano, como variaciones de un único nucleótido (SNV). Para detectar estos biomarcadores se dispone de una amplia oferta de tecnologías. Sin embargo, muchos de los métodos en uso presentan limitaciones como un elevado coste, complejidad, tiempos de análisis largos o requieren de personal y equipamiento especializado, lo que imposibilita su incorporación masiva en la mayoría de los sistemas sanitarios. Por tanto, existe la necesidad de investigar y desarrollar soluciones analíticas que aporten información sobre las variantes genéticas y que se puedan implementar en diferentes escenarios del ámbito de la salud con prestaciones competitivas y económicamente viables. El objetivo principal de esta tesis ha sido desarrollar estrategias innovadoras para resolver el reto de la detección múltiple de variantes genéticas que se encuentran en forma minoritaria en muestras biológicas de pacientes, cubriendo las demandas asociadas al entorno clínico. Las tareas de investigación se centraron en la combinación de reacciones de discriminación alélica con amplificación selectiva de DNA y el desarrollo de sistemas ópticos de detección versátiles. Con el fin de atender el amplio abanico de necesidades, en el primer capítulo, se presentan resultados que mejoran las prestaciones analíticas de la reacción en cadena de la polimerasa (PCR) mediante la incorporación de una etapa al termociclado y de un agente bloqueante amplificando selectivamente las variantes minoritarias que fueron monitorizadas mediante fluorescencia a tiempo real. En el segundo capítulo, se logró la discriminación alélica combinando la ligación de oligonucleótidos con la amplificación de la recombinasa polimerasa (RPA), que al operar a temperatura constante permitió una detección tipo point-of-care (POC). La identificación de SNV se llevó a cabo mediante hibridación en formato micromatriz, utilizando la tecnología Blu-Ray como plataforma de ensayo y detección. En el tercer capítulo, se integró la RPA con la reacción de hibridación alelo especifica en cadena (AS-HCR), en formato array para genotipar SNV a partir de DNA genómico en un chip. La lectura de los resultados se realizó mediante un smartphone. En el último capítulo, se presenta la síntesis de un nuevo reactivo bioluminiscente que se aplicó a la monitorización de biomarcadores de DNA a tiempo real y final de la RPA basada en la transferencia de energía de resonancia de bioluminiscencia (BRET), eliminando la necesidad de una fuente de excitación. Todas las estrategias permitieron un reconocimiento especifico de la variante de interés, incluso en muestras que contenían tan solo 20 copias de DNA genómico diana. Se consiguieron resultados sensibles (límite de detección 0.5% variante/total), reproducibles (desviación estándar relativa < 19%), de manera sencilla (3 etapas o menos), rápida (tiempos cortos de 30-200 min) y permitiendo el análisis simultaneo de varios genes. Como prueba de concepto, estas estrategias se aplicaron a la detección e identificación en muestras clínicas de biomarcadores asociados a cáncer colorrectal y enfermedades cardiológicas. Los resultados se validaron por comparación con los métodos de referencia NGS y PCR, comprobándose que se mejoraban los requerimientos técnicos y la relación coste-eficacia. En conclusión, las investigaciones llevadas a cabo posibilitaron desarrollar herramientas de genotipado con propiedades analíticas competitivas y versátiles, aplicables a diferentes escenarios sanitarios, desde hospitales a entornos con pocos recursos. Estos resultados son prometedores al dar respuesta a la demanda de tecnologías alternativas para el diagnóstico molecular personalizado.[CA] La medicina actual es dirigeix cap a un enfocament més personalitzat basant-se en el diagnòstic molecular del pacient a través de l'estudi de biomarcadors específics. Aplicant aquest principi molecular, el diagnòstic, pronòstic i selecció de la teràpia es recolzen en la identificació de variacions específiques del genoma humà, com variacions d'un únic nucleòtid (SNV). Per a detectar aquests biomarcadors, es disposa d'una àmplia oferta de tecnologies. No obstant això, molts dels mètodes en ús presenten limitacions com un elevat cost, complexitat, temps d'anàlisis llargues o requereixen de personal i equipament especialitzat, la qual cosa impossibilita la seua incorporació massiva en la majoria dels sistemes sanitaris. Per tant, existeix la necessitat d'investigar i desenvolupar solucions analítiques que aporten informació sobre les variants genètiques i que es puguen implementar en diferents escenaris de l'àmbit de la salut amb prestacions competitives i econòmicament viables. L'objectiu principal d'aquesta tesi ha sigut desenvolupar estratègies innovadores per a resoldre el repte de la detecció múltiple de variants genètiques que es troben en forma minoritària en mostres biològiques de pacients, cobrint les demandes associades a l'entorn clínic. Les tasques d'investigació es van centrar en la combinació de reaccions de discriminació al·lèlica amb amplificació selectiva de DNA i al desenvolupament de sistemes òptics de detecció versàtils. Amb la finalitat d'atendre l'ampli ventall de necessitats, en el primer capítol, es presenten resultats que milloren les prestacions analítiques de la reacció en cadena de la polimerasa (PCR) mitjançant la incorporació d'una etapa al termociclat i d'un agent bloquejant amplificant selectivament les variants minoritàries que van ser monitoritzades mitjançant fluorescència a temps real. En el segon capítol, es va aconseguir la discriminació al·lèlica combinant el lligament d'oligonucleòtids amb l'amplificació de la recombinasa polimerasa (RPA), que en operar a temperatura constant va permetre una detecció tipus point-of-care (POC). La identificació de SNV es va dur a terme mitjançant hibridació en format micromatriu, utilitzant la tecnologia Blu-Ray com a plataforma d'assaig i detecció. En el tercer capítol, es va integrar la RPA amb la reacció d'hibridació al·lel específica en cadena (AS-HCR), en format matriu per a genotipar SNV a partir de DNA genòmic en un xip. La lectura dels resultats es va realitzar mitjançant un telèfon intel·ligent. En l'últim capítol, es presenta la síntesi d'un nou reactiu bioluminescent que es va aplicar al monitoratge de biomarcadors de DNA a temps real i final de la RPA basada en la transferència d'energia de ressonància de bioluminescència (BRET), eliminant la necessitat d'una font d'excitació. Totes les estratègies van permetre un reconeixement específic de la variant d'interès, fins i tot en mostres que només contenien 20 còpies de DNA genòmic diana. Es van aconseguir resultats sensibles (límit de detecció 0.5% variant/total), reproduïbles (desviació estàndard relativa < 19%), de manera senzilla (3 etapes o menys), ràpida (temps curts de 30-200 min) i permetent l'anàlisi simultània de diversos gens. Com a prova de concepte, aquestes estratègies es van aplicar a la detecció i identificació en mostres clíniques de biomarcadors associats a càncer colorectal i a malalties cardiològiques. Els resultats es van validar per comparació amb els mètodes de referència NGS i PCR, comprovant-se que es milloraven els requeriments tècnics i la relació cost-eficàcia. En conclusió, les investigacions dutes a terme van possibilitar desenvolupar eines de genotipat amb propietats analítiques competitives i versàtils, aplicables a diferents escenaris sanitaris, des d'hospitals a entorns amb pocs recursos. Aquests resultats són prometedors en donar resposta a la demanda de tecnologies alternatives per al diagnòstic molecular personalitzat.[EN] Current medicine is moving towards a more personalized approach based on the patients' molecular diagnosis through the study of specific biomarkers. Diagnosis, prognosis and therapy selection, applying this molecular principle, rely on identifying specific variations in the human genome, such as single nucleotide variations (SNV). A wide range of technologies is available to detect these biomarkers. However, many of the employed methods have limitations such as high cost, complexity, long analysis times, or requiring specialized personnel and equipment, making their massive incorporation in most healthcare systems impossible. Therefore, there is a need to research and develop analytical solutions that provide information on genetic variants that can be implemented in different health scenarios with competitive and economically feasible performances. The main objective of this thesis has been to develop innovative strategies to solve the challenge of multiple detection of genetic variants that are found in a minority amount in patient samples, covering the demands associated with the clinical setting. Research tasks focused on the combination of allelic discrimination reactions with selective DNA amplification and the development of versatile optical detection systems. In order to meet the wide range of needs, in the first chapter, the analytical performances of the polymerase chain reaction (PCR) were improved by incorporating a thermocycling step and a blocking agent to amplify selectively minority variants that were monitored by real-time fluorescence. In the second chapter, allelic discrimination was achieved by combining oligonucleotide ligation with recombinase polymerase amplification (RPA), which operates at a constant temperature, allowing point-of-care (POC) detection. SNV identification was carried out by hybridization in microarray format, using Blu-Ray technology as the assay platform and detector. RPA was integrated with allele-specific hybridization chain reaction (AS-HCR), in an array format to genotype SNV from genomic DNA on a chip in the third chapter. The reading of the results was performed using a smartphone. In the last chapter, a new bioluminescent reagent was synthesized. It was applied to real-time and endpoint DNA biomarker monitoring based on bioluminescence resonance energy transfer (BRET), eliminating the need for an excitation source. All the strategies allowed specific recognition of the target variant, even in samples containing as few as 20 copies of target genomic DNA. Sensitive (limit of detection 0.5% variant/total), reproducible (relative standard deviation < 19%), simple (3 steps or less), fast (short times of 30-200 min) results were achieved, allowing simultaneous analysis of several genes. As proof of concept, these strategies were applied to detect and identify biomarkers associated with colorectal cancer and cardiological diseases in clinical samples. The results were validated by comparison with reference methods such as NGS and PCR, proving that the technical requirements and cost-effectiveness were improved. In conclusion, the developed research made it possible to develop genotyping tools with competitive analytical properties and versatile, applicable to different healthcare scenarios, from hospitals to limited-resource environments. These results are promising since they respond to the demand for alternative technologies for personalized molecular diagnostics.The authors acknowledge the financial support received from the Generalitat Valenciana PROMETEO/2020/094, GRISOLIA/2014/024 PhD Grant and GVA-FPI-2017 PhD grant, the Spanish Ministry of Economy and Competitiveness MINECO projects CTQ2016-75749-R and PID2019-110713RB-I00 and European Regional Development Fund (ERDF).Lázaro Zaragozá, A. (2022). Study of Strategies for Genetic Variant Discrimination and Detection by Optosensing [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/185216TESISCompendi

Optimization of a microarray-based biosensor for detection of viral pathogens

Rapid and sensitive detection of viral infections is of significant importance for improving patient care and containing outbreaks that threaten public health. Although there has been an enormous effort to develop point-of-care biosensors for viral diagnostics applications, sensitive, robust and easily portable platforms have yet to be realized. This dissertation focuses on optimization of a multiplexed immunoassay platform for viral diagnostics applications using a label-free optical biosensor termed Single-Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). SP-IRIS utilizes an antibody microarray that captures the target viruses and an optical instrument that allows visualization of individual captured virus particles. Since this platform relies on capture of whole viruses, it is crucial to identify high-affinity antibodies that are capable of recognizing intact virions. For this purpose, we screened various antibodies for their performance on the SP-IRIS platform. By screening 43 different antibodies for three different viruses, we demonstrated specific and sensitive detection of different viruses and different subtypes of the same virus. This work allowed us to assemble an antibody microarray capable of multiplexed detection that has been tested in our laboratory as well as at two separate high-containment facilities. Next, we adapted a different antibody immobilization technique, DNA-directed antibody immobilization (DDI), to the SP-IRIS platform as a means to improve the sensitivity and robustness of the assay. First, we characterized the elevation of the antibodies conjugated to a DNA sequence on a three-dimensional polymeric surface using a fluorescence axial localization technique, Spectral Self-Interference Fluorescence Microscopy, determining the optimal length of the DNA linkers for SP-IRIS substrates. We subsequently showed the specific detection of Vesicular Stomatitis Virus (VSV) expressing Ebola glycoprotein on SP-IRIS platform using the DDI approach. We showed that DNA-conjugated antibodies improve the capture efficiency resulting in over a ten-fold improvement in assay sensitivity compared to directly immobilized antibodies. To demonstrate the feasibility of the DDI technique for multiplexed virus detection utilizing SP-IRIS, we used VSVs expressing Ebola, Marburg or Lassa surface glycoproteins and successfully demonstrated specific and multiplexed detection using a DNA microarray surface. We also combined this approach with a passive microfluidic cartridge, demonstrating the feasibility of SP-IRIS as a rapid testing technique that is well suited for point-of-care applications

Multiplexed and Reiterative Detection of Protein Markers in Cells using Dynamic Nucleic Acid Complexes

The diagnosis, staging and clinical management of cancer and other diseases is becoming increasingly reliant upon the identification and quantification of molecular markers as well their spatial distribution in histological samples. Yet, due to spectral overlap of dyes and the inability to remove probes without affecting marker integrity, immunohistological methods are limited by the number of markers that can be examined on a single specimen resulting in loss of information that could be vital to diagnosis or treatment. This dissertation describes the development and characterization of an erasable multi-color imaging technology capable of detecting large numbers of molecular markers on a single biological sample. The system consists of (1) 'targets', which are single or partially hybridized DNA strands conjugated to a protein of interest for biomarker recognition in cells, and (2) multi-strand, fluorophore-containing DNA 'probe complexes' that react with the DNA portion of the target in a sequence dependent fashion to create fluorescent reporting complexes. The addition of a quencher-bearing ssDNA displaces the target's DNA strand to effectively remove the dye from the marker so that the sample can be re-imaged for other markers with minimal interference from prior iii rounds of labeling. Orthogonal DNA sequences and spectrally-separated dyes can be used to create multiple, unique target/probe pairs that associate specifically and can be imaged in parallel. The overall utility of this technology depends on high specificity of targets to respective probe complexes, highly efficient labeling and erasing to ensure that fluorescent signals can be used to fully quantify target abundance without the interference of signals from previous rounds of labeling, and short reaction times to allow for multiple rounds of processing on the same sample without loss of integrity. Based on the above criteria, three classes of probes were designed and their structure-function relationships elucidated to determine the contributions of complex size, free energy differences between intermediate states, and strand displacement on labeling and erasing kinetics and efficiencies on cells. A comparison of the kinetics of the labeling and erasing reactions for the three different constructs showed that reaction efficiencies depend less on calculated net free energy change than on the engineered state of the complex during the strand displacement reaction (i.e., the type of strand displacement reaction it participates in). This new paradigm in probe design allowed the system to meet its design goals, potentially increasing the diagnostic power of individual histological specimens and opening the door to more sophisticated analyses of cell phenotype and its functional relationship to disease

Next-generation sequencing technologies and applications for human genetic history and forensics

Rapid advances in the development of sequencing technologies in recent years have enabled an increasing number of applications in biology and medicine. Here, we review key technical aspects of the preparation of DNA templates for sequencing, the biochemical reaction principles and assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, and bioinformatic approaches for sequence alignment, variant calling and assembly. We also discuss some of the most important advances that the new sequencing technologies have brought to the fields of human population genetics, human genetic history and forensic genetics

Fluorescence-based super-resolution-microscopy strategies for chromatin studies

Super-resolution microscopy (SRM) is a prime tool to study chromatin organisation at near biomolecular resolution in the native cellular environment. With fluorescent labels DNA, chromatin-associated proteins and specific epigenetic states can be identified with high molecular specificity. The aim of this review is to introduce the field of diffraction-unlimited SRM to enable an informed selection of the most suitable SRM method for a specific chromatin-related research question. We will explain both diffraction-unlimited approaches (coordinate-targeted and stochastic-localisation-based) and list their characteristic spatio-temporal resolutions, live-cell compatibility, image-processing, and ability for multi-colour imaging. As the increase in resolution, compared to, e.g. confocal microscopy, leads to a central role of the sample quality, important considerations for sample preparation and concrete examples of labelling strategies applicable to chromatin research are discussed. To illustrate how SRM-based methods can significantly improve our understanding of chromatin functioning, and to serve as an inspiring starting point for future work, we conclude with examples of recent applications of SRM in chromatin research.</p

Using metabarcoding to reveal and quantify plant-pollinator interactions.

12 pagesInternational audienceGiven the ongoing decline of both pollinators and plants, it is crucial to implement effective methods to describe complex pollination networks across time and space in a comprehensive and high-throughput way. Here we tested if metabarcoding may circumvent the limits of conventional methodologies in detecting and quantifying plant-pollinator interactions. Metabarcoding experiments on pollen DNA mixtures described a positive relationship between the amounts of DNA from focal species and the number of trnL and ITS1 sequences yielded. The study of pollen loads of insects captured in plant communities revealed that as compared to the observation of visits, metabarcoding revealed 2.5 times more plant species involved in plant-pollinator interactions. We further observed a tight positive relationship between the pollen-carrying capacities of insect taxa and the number of trnL and ITS1 sequences. The number of visits received per plant species also positively correlated to the number of their ITS1 and trnL sequences in insect pollen loads. By revealing interactions hard to observe otherwise, metabarcoding significantly enlarges the spatiotemporal observation window of pollination interactions. By providing new qualitative and quantitative information, metabarcoding holds great promise for investigating diverse facets of interactions and will provide a new perception of pollination networks as a whole