Academic literature on the topic 'Short read and long read sequencing'
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Journal articles on the topic "Short read and long read sequencing"
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Shumate, Alaina, Brandon Wong, Geo Pertea, and Mihaela Pertea. "Improved transcriptome assembly using a hybrid of long and short reads with StringTie." PLOS Computational Biology 18, no. 6 (June 1, 2022): e1009730. http://dx.doi.org/10.1371/journal.pcbi.1009730.
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Short-read RNA sequencing and long-read RNA sequencing each have their strengths and weaknesses for transcriptome assembly. While short reads are highly accurate, they are rarely able to span multiple exons. Long-read technology can capture full-length transcripts, but its relatively high error rate often leads to mis-identified splice sites. Here we present a new release of StringTie that performs hybrid-read assembly. By taking advantage of the strengths of both long and short reads, hybrid-read assembly with StringTie is more accurate than long-read only or short-read only assembly, and on some datasets it can more than double the number of correctly assembled transcripts, while obtaining substantially higher precision than the long-read data assembly alone. Here we demonstrate the improved accuracy on simulated data and real data from Arabidopsis thaliana, Mus musculus, and human. We also show that hybrid-read assembly is more accurate than correcting long reads prior to assembly while also being substantially faster. StringTie is freely available as open source software at https://github.com/gpertea/stringtie.
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Stapleton, James A., Jeongwoon Kim, John P. Hamilton, Ming Wu, Luiz C. Irber, Rohan Maddamsetti, Bryan Briney, et al. "Haplotype-Phased Synthetic Long Reads from Short-Read Sequencing." PLOS ONE 11, no. 1 (January 20, 2016): e0147229. http://dx.doi.org/10.1371/journal.pone.0147229.
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Nguyen, Son Hoang, Minh Duc Cao, and Lachlan J. M. Coin. "Real-time resolution of short-read assembly graph using ONT long reads." PLOS Computational Biology 17, no. 1 (January 20, 2021): e1008586. http://dx.doi.org/10.1371/journal.pcbi.1008586.
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A streaming assembly pipeline utilising real-time Oxford Nanopore Technology (ONT) sequencing data is important for saving sequencing resources and reducing time-to-result. A previous approach implemented in npScarf provided an efficient streaming algorithm for hybrid assembly but was relatively prone to mis-assemblies compared to other graph-based methods. Here we present npGraph, a streaming hybrid assembly tool using the assembly graph instead of the separated pre-assembly contigs. It is able to produce more complete genome assembly by resolving the path finding problem on the assembly graph using long reads as the traversing guide. Application to synthetic and real data from bacterial isolate genomes show improved accuracy while still maintaining a low computational cost. npGraph also provides a graphical user interface (GUI) which provides a real-time visualisation of the progress of assembly. The tool and source code is available at https://github.com/hsnguyen/assembly.
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Greenman, Noah, Sayf Al-Deen Hassouneh, Latifa S. Abdelli, Catherine Johnston, and Taj Azarian. "Improving Bacterial Metagenomic Research through Long-Read Sequencing." Microorganisms 12, no. 5 (May 4, 2024): 935. http://dx.doi.org/10.3390/microorganisms12050935.
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Metagenomic sequencing analysis is central to investigating microbial communities in clinical and environmental studies. Short-read sequencing remains the primary approach for metagenomic research; however, long-read sequencing may offer advantages of improved metagenomic assembly and resolved taxonomic identification. To compare the relative performance for metagenomic studies, we simulated short- and long-read datasets using increasingly complex metagenomes comprising 10, 20, and 50 microbial taxa. Additionally, we used an empirical dataset of paired short- and long-read data generated from mouse fecal pellets to assess real-world performance. We compared metagenomic assembly quality, taxonomic classification, and metagenome-assembled genome (MAG) recovery rates. We show that long-read sequencing data significantly improve taxonomic classification and assembly quality. Metagenomic assemblies using simulated long reads were more complete and more contiguous with higher rates of MAG recovery. This resulted in more precise taxonomic classifications. Principal component analysis of empirical data demonstrated that sequencing technology affects compositional results as samples clustered by sequence type, not sample type. Overall, we highlight strengths of long-read metagenomic sequencing for microbiome studies, including improving the accuracy of classification and relative abundance estimates. These results will aid researchers when considering which sequencing approaches to use for metagenomic projects.
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Craddock, Hillary A., Yair Motro, Bar Zilberman, Boris Khalfin, Svetlana Bardenstein, and Jacob Moran-Gilad. "Long-Read Sequencing and Hybrid Assembly for Genomic Analysis of Clinical Brucella melitensis Isolates." Microorganisms 10, no. 3 (March 14, 2022): 619. http://dx.doi.org/10.3390/microorganisms10030619.
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Brucella melitensis is a key etiological agent of brucellosis and has been increasingly subject to characterization using sequencing methodologies. This study aimed to investigate and compare short-read, long-read, and hybrid assemblies of B. melitensis. Eighteen B. melitensis isolates from Southern Israel were sequenced using Illumina and the Oxford Nanopore (ONP) MinION, and hybrid assemblies were generated with ONP long reads scaffolded on Illumina short reads. Short reads were assembled with INNUca with SPADes, long reads and hybrid with dragonflye. Abricate with the virulence factor database (VFDB) and in silico PCR (for the genes BetB, BPE275, BSPB, manA, mviN, omp19, perA, PrpA, VceC, and ureI) were used for identifying virulence genes, and a total of 61 virulence genes were identified in short-read, long-read, and hybrid assemblies of all 18 isolates. The phylogenetic analysis using long-read assemblies revealed several inconsistencies in cluster assignment as compared to using hybrid and short-read assemblies. Overall, hybrid assembly provided the most comprehensive data, and stand-alone short-read sequencing provided comparable data to stand-alone long-read sequencing regarding virulence genes. For genomic epidemiology studies, stand-alone ONP sequencing may require further refinement in order to be useful in endemic settings.
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Botton, Mariana R., Yao Yang, Erick R. Scott, Robert J. Desnick, and Stuart A. Scott. "Phased Haplotype Resolution of the SLC6A4 Promoter Using Long-Read Single Molecule Real-Time (SMRT) Sequencing." Genes 11, no. 11 (November 12, 2020): 1333. http://dx.doi.org/10.3390/genes11111333.
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The SLC6A4 gene has been implicated in psychiatric disorder susceptibility and antidepressant response variability. The SLC6A4 promoter is defined by a variable number of homologous 20–24 bp repeats (5-HTTLPR), and long (L) and short (S) alleles are associated with higher and lower expression, respectively. However, this insertion/deletion variant is most informative when considered as a haplotype with the rs25531 and rs25532 variants. Therefore, we developed a long-read single molecule real-time (SMRT) sequencing method to interrogate the SLC6A4 promoter region. A total of 120 samples were subjected to SLC6A4 long-read SMRT sequencing, primarily selected based on available short-read sequencing data. Short-read genome sequencing from the 1000 Genomes (1KG) Project (~5X) and the Genetic Testing Reference Material Coordination Program (~45X), as well as high-depth short-read capture-based sequencing (~330X), could not identify the 5-HTTLPR short (S) allele, nor could short-read sequencing phase any identified variants. In contrast, long-read SMRT sequencing unambiguously identified the 5-HTTLPR short (S) allele (frequency of 0.467) and phased SLC6A4 promoter haplotypes. Additionally, discordant rs25531 genotypes were reviewed and determined to be short-read errors. Taken together, long-read SMRT sequencing is an innovative and robust method for phased resolution of the SLC6A4 promoter, which could enable more accurate pharmacogenetic testing for both research and clinical applications.
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Volden, Roger, Theron Palmer, Ashley Byrne, Charles Cole, Robert J. Schmitz, Richard E. Green, and Christopher Vollmers. "Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA." Proceedings of the National Academy of Sciences 115, no. 39 (September 10, 2018): 9726–31. http://dx.doi.org/10.1073/pnas.1806447115.
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High-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-distance information is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. While long-read sequencing technology can provide this long-distance information, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here, we introduce and validate a long-read ONT-based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single-cell samples.
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Iyer, Shruti V., Sara Goodwin, and William Richard McCombie. "Leveraging the power of long reads for targeted sequencing." Genome Research 34, no. 11 (November 2024): 1701–18. http://dx.doi.org/10.1101/gr.279168.124.
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Long-read sequencing technologies have improved the contiguity and, as a result, the quality of genome assemblies by generating reads long enough to span and resolve complex or repetitive regions of the genome. Several groups have shown the power of long reads in detecting thousands of genomic and epigenomic features that were previously missed by short-read sequencing approaches. While these studies demonstrate how long reads can help resolve repetitive and complex regions of the genome, they also highlight the throughput and coverage requirements needed to accurately resolve variant alleles across large populations using these platforms. At the time of this review, whole-genome long-read sequencing is more expensive than short-read sequencing on the highest throughput short-read instruments; thus, achieving sufficient coverage to detect low-frequency variants (such as somatic variation) in heterogenous samples remains challenging. Targeted sequencing, on the other hand, provides the depth necessary to detect these low-frequency variants in heterogeneous populations. Here, we review currently used and recently developed targeted sequencing strategies that leverage existing long-read technologies to increase the resolution with which we can look at nucleic acids in a variety of biological contexts.
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Wick, Ryan R., Louise M. Judd, and Kathryn E. Holt. "Assembling the perfect bacterial genome using Oxford Nanopore and Illumina sequencing." PLOS Computational Biology 19, no. 3 (March 2, 2023): e1010905. http://dx.doi.org/10.1371/journal.pcbi.1010905.
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A perfect bacterial genome assembly is one where the assembled sequence is an exact match for the organism’s genome—each replicon sequence is complete and contains no errors. While this has been difficult to achieve in the past, improvements in long-read sequencing, assemblers, and polishers have brought perfect assemblies within reach. Here, we describe our recommended approach for assembling a bacterial genome to perfection using a combination of Oxford Nanopore Technologies long reads and Illumina short reads: Trycycler long-read assembly, Medaka long-read polishing, Polypolish short-read polishing, followed by other short-read polishing tools and manual curation. We also discuss potential pitfalls one might encounter when assembling challenging genomes, and we provide an online tutorial with sample data (github.com/rrwick/perfect-bacterial-genome-tutorial).
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Eisenstein, Michael. "Startups use short-read data to expand long-read sequencing market." Nature Biotechnology 33, no. 5 (May 2015): 433–35. http://dx.doi.org/10.1038/nbt0515-433.
Full textDissertations / Theses on the topic "Short read and long read sequencing"
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Soundiramourtty, Abirami. "Exploring the transpositional landscape and recent transposable element activity in beech trees using long read mobilome and genome sequencing and with new computational tools." Electronic Thesis or Diss., Perpignan, 2024. http://www.theses.fr/2024PERP0043.
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L’adaptation des organismes aux changements environnementaux est devenue une question fondamentale de la recherche, en particulier face aux impacts du réchauffement climatique. Un axe clé de recherche consiste à comprendre comment les éléments génétiques sous jacent, tels que les éléments transposables (ET). Les ET sont des séquences d'ADN répétés présentes chez tous les Eucaryotes, possédant la capacité unique de se déplacer au sein du génome, un phénomène appelé transposition active. Ainsi, ils peuvent provoquer des mutations en générant des insertions polymorphiques d'ET (TIPs) entre individus, voire des insertions somatiques. En général, les ET restent inactifs grâce à des mécanismes épigénétiques qui limitent leur prolifération incontrôlée. Cependant, ils peuvent être réactivés par divers stimuli environnementaux, rendant la transposition active relativement rare. Cette mobilité des ET peut être révélée en utilisant l'ADN circulaire extrachromosomique (ADNecc) comme marqueur de transposition. Le paysage transpostionnel des TEs et leur activité récente ont été décrits chez des organismes modèles, mais restent inexploités chez les espèces pérennes comme les arbres. Cette étude vise à explorer l’activité transpositionelle récente et la mobilité en cours des ET chez des espèces pérennes non modèles en utilisant le hêtre européen (Fagus sylvatica) comme notre modèle d’étude. Nous avons cherché à étudier l'activité récente des ET et leur mobilité continue en identifiant les variants causés par les ET au sein d'une population et chez un individu (à l'échelle somatique) en utilisant le séquençage du génome complet (WGS) et le séquençage du mobilome (ou ADNecc). Nous avons réalisé le séquençage WGS et du mobilome d'arbres de la forêt de Verzy, connue pour abriter des hêtres nains et tortillards, également appelés « mutants ». Ces arbres présentent des traits morphologiques instables, avec chez certains arbres de nouvelles branches qui se développent avec une forme normale. Nous avons identifié deux ET appartenant au type des Miniature Inverted Repeats Transposable Elements (MITEs), nommés SQUIRREL1 et SQUIRREL2, qui se mobilisent activement dans ces arbres, produisant une grande quantité dADNecc et causant même des variations somatiques. SQUIRREL1 et SQUIRREL2 sont également actifs dans les hêtres de la forêt de la Massane. De plus, dans tous ces arbres, plusieurs d’autres ET, principalement des MITEs, produisent une grande quantité dADNecc, bien que leur niveau d’activité semble varier en fonction des tissus, suggérant que l'activité des ET varie selon le stade de développement et indiquant une transposition dominée par les MITEs chez le hêtre. Parallèlement, nous avons étudié les TIPs dans une population de hêtres de la forêt de la Massane, une forêt ancienne classée au patrimoine mondial de l'UNESCO. En séquençant 150 arbres, nous avons cherché à comprendre comment les ET contribuent à la diversité génétique de l'ensemble de la population en détectant les TIPs générés par les Long Terminal Repeats rétrotransposons (LTR RT) et les MITEs en utilisant le séquençage WGS. Nous avons détecté environ 30 000 TIPs de LTR-RT chez chaque individu, contre 70 000 TIPs de MITEs. La plupart de ces TIPs restent à faible fréquence mais de nombreux MITE-TIPs restent localisés près de gènes fonctionnels et conservés au sein de la population. À partir des TIPs, nous avons identifié plusieurs points chauds de variation et des régions conservées le long du génome du hêtre permettant d’abordant la structuration du génome chez cette espèce. Pour conclure, notre étude met en lumière l’importance des ET dans la structuration du paysage génomique des arbres, en particulier dans la manière dont ces éléments contribuent à l’évolution des espèces à longue durée de vie. Les recherches futures pourraient étendre ces travaux à d’autres espèces d'arbres et explorer si les schémas observés se retrouvent dans d’autres espèces d’arbresThe adaptation of organisms to environmental changes has become a fundamental research question,particularly in the context of climate change. A key area of this research is to identify underlying genetic elements, such as transposable elements (TEs), contributing to this process. TEs are repetitive DNA sequences found across all eukaryotes, possessing the unique ability to move within the genome, a phenomenon known as active transposition. They can cause mutations by generating transposable element insertion polymorphisms (TIPs) between individuals, and even somatic insertions. Generally, TEs remain inactive by epigenetic mechanisms that limit their uncontrolled proliferation. However, they can be reactivated upon various environmental stimuli, making active transposition relatively rare. TE mobility can be detected using extrachromosomal circular DNA (eccDNA) as a marker of transposition. The transpositional landscape of TEs and their recent activity have been documented in model organisms but remain underexplored in perennial species such as trees. This study aims to investigate recent transpositional activity and ongoing mobility of TEs in non-model perennial species, using European beech (Fagus sylvatica) as our model. We sought to study recent TE activity and their continuous mobility byidentifying TE-induced variants within a population and in an individual (at the somatic scale) using whole-genome sequencing (WGS) and mobilome sequencing (eccDNA). We conducted WGS and mobilome sequencing of trees from the Verzy forest, known for its dwarf and tortuous beeches, also referred as "mutants." These trees exhibit unstable phenotypical traits, with some trees developing new normal branches. We identified two TEs belonging to the Miniature Inverted Repeat Transposable Elements (MITEs) type, named SQUIRREL1 and SQUIRREL2, which are actively mobilizing in these trees, producing large amounts of eccDNA and even causing somatic variations.SQUIRREL1 and SQUIRREL2 are also active in beech trees from the Massane forest. Furthermore, in all these trees, several other TEs,mainly MITEs, produce significant amounts of eccDNA, although their activity levels appear to vary depending on the tissues, suggesting that TE activity could be tissue-specific indicating MITE-dominated transposition in beech. Simultaneously, we investigated TIPs in a population of beech trees from the Massane forest, an ancient forest classified as a UNESCO World Heritage site. By sequencing 150 trees, we aimed to understand how TEs contribute to the genetic diversity of the entire population by detecting TIPs generated by Long Terminal Repeat retrotransposons (LTR-RTs) and MITEs using WGS. We detected approximately 30,000 LTR-RT TIPs in each individual, compared to 70,000 MITE TIPs. While most of these TIPs remain at low frequency, many MITE-TIPs are located near functional genes and more conserved within the population. Using these TIPs, we identified several hotspots of variation and conserved regions along the beech genome, providing insights into genome structure in this species. In conclusion, our study highlights the importance of TEs in shaping the genomic landscape of trees, particularly in understanding how these elements contribute to the evolution of long-lived species. Future research could expand this work to other tree species and explore whether the patterns observed in beeches are common in other types of trees
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Whiteford, Nava. "String matching in DNA sequences : implications for short read sequencing and repeat visualisation." Thesis, University of Southampton, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.438668.
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Chacon, de San Baldomero Alejandro. "Read mapping on heterogeneous systems: scalability strategies for bioinformatic primitives." Doctoral thesis, Universitat Autònoma de Barcelona, 2021. http://hdl.handle.net/10803/671736.
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La seqüenciació genòmica és un component clau en nous avenços en medicina, i la seva democratització és un pas important per millorar l’accessibilitat per al pacient. Els beneficis implícits en el descobriment de noves variants genètiques són molt amplis, incloent des de la detecció precoç de càncer com la medicina personalitzada, passant pel disseny de fàrmacs i l’edició genòmica. Tots aquests usos potencials han incrementat exponencialment l’interès de la comunitat científica en el camp de la bioinformàtica durant els últims anys. A més, el sorgiment dels mètodes de Seqüenciació de Nova Generació ha contribuït a la reducció ràpida dels costos de seqüenciació, permetent el desenvolupament de noves aplicacions genòmiques. El principal objectiu d’aquesta tesi és el de millorar el rendiment i precisió de l’estat de l’art de la seqüenciació genètica a través de l’ús de plataformes de còmput heterogeni i sistemes de computació híbrida. Més específicament, el treball s’ha centrat en l’acceleració de el problema de mapeig de reads curts, ja que es descriu com un dels estadis del pipeline amb un major cost computacional. De forma global, s’ aspirava a reduir el temps de processament i el cost de la seqüenciació genètica, incrementant la disponibilitat d’aquest tipus d’anàlisi. La principal contribució d’aquesta tesi és la integració GPU del mapper GEM3 (GEM3-GPU). Aquest mapper reporta les mateixes dades de sortida per CPU i GPU, i és un dels primers mappers GPU que permet l’alineament de reads llargs i variables. Les propostes han estat validades utilitzant dades reals, ja que el mapper ha estat corrent en producció en un centre de seqüenciació genòmica (Centre Nacional d’Anàlisi Genòmica (CNAG)). En conjunció amb el mapper GEM3-GPU, durant aquesta tesi s’ha creat una llibreria bioinformàtica en CUDA (GEM-cutter). La llibreria aporta blocs de primitives GPU bàsiques que han estat altament optimitzades. Gem-cutter ofereix una API basada en primitives send and receive (message passing), i incorpora un scheduler per balancejar el treball. A més, la llibreria suporta totes les arquitectures GPU i Multi-GPU.La secuenciación genómica es un componente clave en nuevos avances en medicina, y su democratización es un paso importante hacia la accesibilidad para el paciente. Los beneficios implícitos en el descubrimiento de nuevas variantes genéticas son muy amplios, incluyendo desde la detección precoz de cáncer como la medicina personalizada, pasando por el diseño de fármaco y la edición genómica. Estos usos potenciales han incrementado exponencialmente el interés de la comunidad científica en el campo de la bioinformática durante los últimos años. Además, el surgimiento de los métodos de Secuenciación de Nueva Generación ha contribuido a la reducción rápida de los costes de secuenciación, permitiendo el desarrollo de nuevas aplicaciones genómicas. El principal objetivo de esta tesis es el de mejorar el rendimiento y precisión del estado del arte de la secuenciación genética a través del uso de plataformas de computo heterogéneo y sistemas de hardware híbridos. Más específicamente, el trabajo se ha centrado en la aceleración del problema del short-read mapping, dado que se describe como uno de los estadíos del pipeline con un mayor coste computacional. De forma global, se aspiraba a reducir el tiempo de procesado y el coste de la secuenciación genética, incrementando su disponibilidad. La principal contribución de esta tesis es la integración GPU del mapper GEM3 (GEM3-GPU). Este mapper reporta los mismos datos de salida para CPU y GPU, y es uno de los primeros mappers GPU que permite el alineamiento de reads largos y variables. Las propuestas han sido validadas utilizando datos reales, dado que el mapper ha estado corriendo en producción en un centro de secuenciación (Centro Nacional de Análisis Genómico (CNAG)). En conjunción con el mapper GEM3-GPU, durante esta tesis se ha creado una librería bioinformática en CUDA (GEM-cutter). La librería provee bloques de primitivas GPU básicas que han sido altamente optimizadas. Gem-cutter ofrece una API basada en primitivas de send and receive (message passing), e incorpora un scheduler para balancear el trabajo. Además, la librería soporta todas las arquitecturas GPU y Multi-GPU.
Genomic sequencing is the key component of new advances in medicine, and its democratization is an important step in improving accessibility for the patient. The benefits involved in discovering new genomic variations are vast and include everything from early cancer detection to personalized medicine, drug design and genome editing. All of these potential uses have greatly increased the interest of the scientific community in the field of bioinformatics in recent years. Moreover, the emergence of next-generation sequencing methods has contributed to the rapid reduction of sequencing costs, enabling new applications of genomics in precision medicine. The main goal of this thesis is to improve the state of the art in performance and accuracy for genome sequencing through the use of heterogeneous computing platforms and hybrid hardware systems. More specifically, the work is focused on accelerating the problem of short-read mapping, as it is described as one of the most computationally expensive parts of the pipeline process. Overall, we aim to reduce the processing time and cost of genome sequencing, and then increasing the availability of this type analysis. The main contribution of this thesis is the full GPU integration of the GEM3 mapper (GEM3-GPU), reporting significant improvements in performance and competitive accuracy results. The mapper reports the same output files for CPU and GPU and is one of the first GPU mappers to allow very long and variable read alignment. The proposals have been validated using real data, since the mapper has been running in production at a genomic sequencing center (Centro Nacional de Análisis Genómico (CNAG)). Together with the GEM3-GPU mapper, a complete bioinformatics CUDA library (GEM-cutter) has been created. The library provides the basic building blocks for genomic applications, which are highly optimised to run on GPUs. Gem-cutter offers an API based on send and receive primitives (message passing) and incorporates a scheduler to balance the work. Furthermore, the library supports all GPU architectures and Multi-GPU execution.
Universitat Autònoma de Barcelona. Programa de Doctorat en Informàtica
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Targon, Robin. "A novel method for the production of long DNA sequences from short reads." Doctoral thesis, Università degli studi di Padova, 2015. http://hdl.handle.net/11577/3424278.
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Next Generation Sequencing (NGS) has deeply changed our way to study genome biology: in the last ten years an astonishing amount of evidence ranging from the transcriptome variability to the association patterns of specific proteins with DNA or RNA sequences were produced with this technology, thus opening the way to amazing discoveries and perspectives.
Unfortunately, the short length of the sequencing reads produced by second generation sequencers is limiting the potential of this technology. In particular some very interesting studies have been hampered by the short read length. High-quality long reads would permit much better approaches to full-length transcripts analysis, alternative splicing, RNA editing, de novo whole genome assembly, genomic structural variations and haplotype characterization.
The study that I conducted for my doctorate focused on the possibility to produce high-quality long reads using NGS technology. The first motivation behind the development of this project was to investigate full-length transcripts and in particular to verify the hypothesis whether the pattern of alternative splicing could be associated to transcription start sites. A further motivation was the application of this technology to de novo whole genome assembly.
Since at the instrumental level the limits of the read length is not amendable, I addressed my efforts towards the development of a method to reconstruct the sequence of long DNA or RNA molecules by precise local assembly of short reads produced by second generation sequencers.
The idea that I wanted to exploit is based on “molecular barcoding”. Typically, barcodes are short DNA sequence tags that are included in the adaptors and used for the preparation of NGS libraries. Barcodes make possible the association of each read to its corresponding library, allowing the analysis of multiple samples in the same sequencing run. In my project I used barcodes for a very different purpose. In fact, my objective was to label individual DNA or RNA molecules with univocal barcodes, to enable the identification of all the reads generated from the subfragments of each original molecule. For this purpose I used random barcodes, considering that reads with the same barcode would come from the same original DNA/RNA molecule. Therefore, in comparison to standard barcoding techniques, my approach has two main differences: firstly it is a single molecule barcoding, secondly the barcodes are made by random sequences.
A considerable part of my work was dedicated to the development of reliable genetic engineering strategies to obtain mate-pair libraries constituted on one side by the barcoded end and on the other side by a random region of the original DNA or RNA molecule. Every step of the protocol was carefully optimized in order to make the method simple and at the same time robust.
Several trials were performed to test the method. Although in these trials we limited the analysis to a low coverage, we found that mate pair reads sharing the same barcode were mostly mapping in clustered genomic positions, as expected. Our results, albeit preliminary, demonstrate that the method so far developed is capable to work.
Although some steps of the protocol could be further optimized, the method is now applied to produce long genomic reads with high coverage. Furthermore, some adaptations are now implemented to apply the method also to transcriptome samples.L'avvento dei sequenziatori di ultima generazione (NGS) ha profondamente cambiato il nostro approccio allo studio del genoma e dell'espressione genica: negli ultimi dieci anni è stata prodotta un'incredibile quantità di dati e di evidenze sperimentali riguardanti la complessità del trascrittoma e le interazioni tra specifiche proteine e molecole di DNA o RNA, aprendo così la strada ad entusiasmanti scoperte ed applicazioni tecnologiche. Sfortunatamente, la ridotta lunghezza delle sequenze prodotte dai sequenziatori di seconda generazione limita le potenzialità di questa tecnologia. Nello specifico, alcune interessanti applicazioni quali l'analisi degli splicing alternativi e dell'RNA-editing, l'assemblaggio di genomi ex novo, la caratterizzazione di aplotipi e l'identificazione di variazioni strutturali a livello genomico, beneficerebbero sicuramente di una tecnologia in grado di produrre lunghe sequenze ad alta qualità. Lo studio che ho condotto durante il mio dottorato di ricerca è stato finalizzato alla produzione di lunghe sequenze ad alta qualità utilizzando gli attuali sequenziatori di seconda generazione. La principale motivazione che ha guidato questo studio è stata la volontà di caratterizzare a livello di sequenza nucleotidica le diverse isoforme trascrizionali in modo da poter verificare l'ipotesi di una relazione funzionale tra l'utilizzo di specifici siti d'inizio trascrizione e lo splicing alternativo degli esoni. Un'ulteriore motivazione era rappresentata dalla possibilità di ottenere la sequenza di lunghi frammenti di DNA al fine di facilitare l'assemblaggio di genomi. Non essendo possibile intervenire sulla lunghezza delle sequenze prodotte dai sequenziatori di seconda generazione, ho sviluppato una strategia che permette di ottenere lunghe sequenze nucleotidiche mediante un preciso assemblaggio di sequenze corte derivanti da una singola molecola. Questa strategia si basa sul concetto di “barcoding” molecolare. Un “barcode”, letteralmente “codice a barre”, è un corto frammento di DNA a sequenza nucleotidica nota che viene aggiunto a tutte le molecole di uno specifico campione. In questo modo è possibile sequenziare diversi campioni simultaneamente e associare ogni sequenza al proprio campione di provenienza semplicemente leggendo il “barcode” ad essa associato. Nel mio progetto lo scopo e la natura dei “barcode” è differente: i “barcode” utilizzati hanno sequenza casuale, in moda da poter marcare ogni singola molecola del campione con una sequenza univoca. La presenza di un “barcode” univoco permette l'assegnazione delle sequenze prodotte alla molecola di origine e, quindi, il loro corretto assemblaggio. Una parte considerevole di questo lavoro è stata dedicata allo sviluppo di strategie di ingegneria genetica che permettessero la costruzione di librerie “mate pair” in cui parte della sequenza fosse costituita dal “barcode”, mentre l'altra parte rappresentasse una porzione casuale della molecola di DNA o RNA di origine. Ogni singolo passaggio del protocollo è stato ottimizzato al fine di rendere il metodo più semplice e robusto. Diverse prove di sequenziamento sono state effettuate per poter valutare l'efficienza della metodica; sebbene l'analisi di queste prove sia stata condizionata dal basso “coverage” di sequenziamento, abbiamo dimostrato come le sequenze “mate pair” che condividono lo stesso “barcode” si allineino, come atteso, a livello della stessa posizione genomica. I risultati ottenuti, sebbene siano preliminari, dimostrano che il metodo sviluppato funziona. Nonostante alcuni passaggi del protocollo richiedano un'ulteriore ottimizzazione, il metodo verrà a breve impiegato per la produzione di lunghe sequenze genomiche aumentando il “coverage” di sequenziamento. Nel prossimo futuro l'introduzione di alcune modifiche minori al protocollo permetterà di estendere il suo utilizzo all'analisi di trascrittomi.
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Long, Evan Michael. "Genomic Structural Variation Across Five Continental Populations of Drosophila melanogaster." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7335.
Full textAbstract:
Chromosomal structure variations (SV) including insertions, deletions, inversions, and translocations occur within the genome and can have a significant effect on organismalphenotype. Some of these effects are caused by structural variations containing genes. Modern sequencing using short reads makes the detection of large structural variations (> 1kb) very difficult. Large structural variations represent a significant amount of the genetic diversity within a population. We used a global sampling of Drosophila melanogaster (Ithaca, Zimbabwe, Beijing, Tasmania, and Netherlands) to represent diverse populations. We used long-read sequencing and optical mapping technologies to identify SVs in these genomes. Because the average read length used for these approaches are much longer than traditional short read sequencing, these maps facilitate the identification of chromosomal SVs of greater size and with more clarity. We found a wide diversity of structural variations in each of the five strains. These structural variations varied greatly in size and location, and significantly affected exonic regions of the genome. Structural variations accounted for a much larger difference in number of base pairs between strains than single nucleotide polymorphisms (SNPs).
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Fuente, Lorente Lorena de la. "Development of a bioinformatics approach for the functional analysis of alternative splicing." Doctoral thesis, Universitat Politècnica de València, 2019. http://hdl.handle.net/10251/124974.
Full textAbstract:
[ES] Uno de los aspectos más apasionantes de la transcripción es la plasticidad transcriptómica y proteómica mediada por los procesos de regulación post-transcripcional (PTR). Los mecanismos PTR como el splicing alternativo (AS) y la poliadenilación alternativa (APA) han emergido como procesos estrechamente regulados que juegan un papel clave en la generación de la complejidad transcriptómica y están asociados con la coordinación de la diferenciación celular o el desarrollo de tejidos. Sin embargo nuestro conocimiento sobre cómo estos mecanismos regulan las propiedades de los productos resultantes para definir el fenotipo es aún muy reducido. La cantidad de variantes existentes y el amplio rango de posibles consecuencias funcionales, hacen su validación funcional una tarea impracticable si se realiza caso por caso. Además, la falta de herramientas para la evaluación funcional orientada a isoformas ha provocado que gran parte del trabajo computacional haya empleado pipelines ad-hoc aplicadas a sistemas biológicos específicos o simplemente hayan confiado en análisis de enriquecimiento GO, los cuales no son informativos del impacto en las propiedades de las isoformas que hay detrás de la regulación PTR.
De hecho, a pesar de las más de sesenta mil publicaciones relativas al AS, muy pocas isoformas se han asociado con propiedades específicas, mientras que el número de nuevas variantes AS/APA con function desconocida crece exponencialmente debido a las técnicas de secuenciación de segunda generación (NGS). Además, y debido a limitaciones técnicas de las NGS para reconstruir la estructura de los transcritos, las tecnologías de secuenciación de tercera generación (TGS) están definiendo una nueva era en la que, por primera vez, es posible conocer la secuencia de elementos estructurales y funcionales en los mRNAs.
En esta tesis se han abordado tres propósitos principales para poder avanzar en el estudio funcional de las isoformas. En primer lugar, con las TGS siendo cada vez más utilizadas, la evaluación de la calidad de los transcriptomas \textit{de novo} es esencial para asegurar la fiabilidad de la diversidad transcriptómica encontrada. La falta de análisis de calidad orientados a secuencias largas ha motivado el desarrollo de SQANTI, una pipeline automatizado para la exhaustiva evaluación de TGS transcriptomas. En segundo lugar, la información a nivel de gen de la mayoría de bases de datos funcionales sigue siendo el principal escollo para el estudio de la variabilidad entre isoformas, especialmente en el caso de las isoformas nuevas, en las que las bases de datos estáticas impiden su caracterización. Así, hemos diseñado IsoAnnot, que construye una base de datos de anotaciones funcionales con resolución a nivel de isoformas integrando información diseminada por múltiples bases de datos y métodos de predicción. Finalmente, la indisponibilidad de métodos para estudiar el impacto funcional de la regulación de isoformas, nos ha motivado a desarrollar tappAS, una herramienta dinámica, flexible y diseñada para facilitar el abordaje de este tipo de estudios.
Por lo tanto, durante esta tesis hemos desarrollado una infraestructura que resuelve los retos principales del análisis funcional de isoformas, proporcionando un conjunto de nuevos métodos y herramientas que ofrecen una oportunidad única para explorar cómo el fenotipo se especifica post-transcripcionalmente, mediante la alteración de las propiedades funcionales de las isoformas expresadas. La aplicación de nuestro análisis a un doble sistema de diferenciación neuronal en ratón definió el efecto de la regulación de isoformas entre la diferenciación de motoneuronas y oligodendrocitos para múltiples elementos funcionales. Entre ellos, hemos descubierto regiones transmembrana que son diferencialmente incluidas en las isoformas expresadas entre ambos tipos celulares y cuya regulación podría estar contribuyendo al control de[CAT] Un dels aspectes més emocionants de la biologia del transcriptoma és l'adaptabilitat contextual de transcriptomes i proteomes eucariotes mitjançant la regulació post-transcripcional (PTR). Els mecanismes PTR, com el splicing alternatiu (AS) i la poliadenilació alternativa (APA), s'han convertit en processos molt regulats que juguen un paper clau en la generació de la complexitat del transcriptoma i en la coordinació de la diferenciació cel·lular o del desenvolupament de teixits. No obstant això, el nostre coneixement de com aquests mecanismes imprimeixen característiques funcionals diferents al conjunt resultant d'isoformes per definir el fenotip observat és encara escàs. El nombre de variants de PTR i les seues conseqüències potencialment funcionals fa que la validació funcional sigui una tasca poc pràctica si es fa cas per cas. A més, la manca d'enfocaments funcionals orientats a isoformes ha fet que gran part del treballs computacionals per esbrinar qüestions funcionals a nivell de transcriptoma siguen estratègies computacionals ad hoc aplicades a sistemes biològics específics o bé basats en un simple anàlisi d'enriquiment GO, que no aporten informació sobre l'impacte de la PTR sobre les propietats de les isoformes. Així, malgrat les més de 60.000 publicacions existents sobre AS, poques de les isoformes existents s'han associat a propietats específiques, mentre que el nombre de noves variants AS/APA amb funcions desconegudes i fins i tot inexplorades augmenta de manera exponencial gràcies a la seqüenciació de nova generació (NGS). A causa de les limitacions tècniques del NGS per reconstruir l'estructura dels transcrits, la seqüenciació d'alt rendiment de transcrits de longitud completa mitjançant tecnologies de tercera generació (TGS) obre una nova era en la transcriptòmica, ja que millora la definició dels models genètics i, per primera vegada, permet associar amb precisió esdeveniments funcionals dins de la molècula d'ARN. Aquesta tesi aborda tres grans reptes per a progressar en l'estudi de la funció de les isoformes. En primer lloc, amb l'aparició i la popularitat creixent del TGS, la definició precisa i la caracterització completa dels transcriptomes de novo són essencials per garantir la qualitat de qualsevol conclusió sobre la diversitat del transcriptoma. La manca d'anàlisis de qualitat orientats a lectures llargues va motivar el desenvolupament de SQANTI (https://bitbucket.org/ ConesaLab / sqanti), una estratègia computacional automatitzada per a la caracterització estructural i l'avaluació de la qualitat dels transcriptomes de longitud completa. En segon lloc, els recursos funcionals existents centrats en el gen suposen una gran limitació per a l'estudi extensiu de la variabilitat funcional de les isoformes, especialment en les noves isoformes, que no es poden caracteritzar per bases de dades estàtiques. Per tant, vam dissenyar IsoAnnot, que construeix dinàmicament una base de dades amb anotacions funcionals a nivell d'isoforma, que utilitza com a informació d'entrada les seqüències dels transcrits i integra informació de diverses bases de dades i mètodes de predicció. Finalment, com no hi havia cap mètode per interrogar l'impacte funcional del PTR, vam desenvolupar nous enfocaments i eines fàcils d'utilitzar, com ara tappAS (http://tappas.org/), dissenyada per facilitar als investigadors els estudis funcionals de transcriptoma complet i de regulació d'isoformes en contexts específics. Per tant, aquesta tesi descriu el desenvolupament d'un marc d'anàlisi que aborda els reptes fonamentals de l'anàlisi funcional d'isoformes. Aplicada a un sistema de diferenciació neuronal murina, vam descobrir regions transmembrana específiques d'isoformes, la modulació de les quals per PTR podria contribuir a controlar la dinàmica mitocondrial específica del tipus cel·lular durant la determinació del destí neuronal.
[EN] One of the most exciting aspects of transcriptome biology is the contextual adaptability of eukaryotic transcriptomes and proteomes by post-transcriptional regulation (PTR). PTR mechanisms such as alternative splicing (AS) and alternative polyadenylation (APA) have emerged as tightly regulated processes playing a key role in generating transcriptome complexity and coordinating cell differentiation or tissue development. However, how these mechanisms imprint distinct functional characteristics on the resulting set of isoforms to define the observed phenotype remains poorly understood. The number of PTR variants and their resulting range of potentially functional consequences makes their functional validation an impractical task if done on a case-by-case basis. Besides, the lack of isoform-oriented functional profiling approaches has made that much of the computational work done to elucidate transcriptome-wide functional questions has either involved ad hoc computational pipelines applied to specific biological systems or has relied on simple GO-enrichment analysis that are not informative about the PTR impact on isoform properties. Thus, even though more than 60,000 publications on AS, a few number of existing isoforms have been associated with specific properties while the number of novel AS/APA variants with unknown and even unexplored functions is exponentially increasing thanks to the use of next-generation sequencing (NGS). Due to the technical limitations of NGS to reconstruct the transcript structure, high-throughput sequencing of full-length transcripts using third-generation technologies (TGS) is opening up a new transcriptomics era that enhances the definition of gene models and, for the first time, enables to precisely associate functional events within the RNA molecule. This thesis addresses three major challenges to the progression of the study of isoform function. First, with the emergence and increasing popularity of TGS, the accurate definition and comprehensive characterisation of de novo transcriptomes is essential to ensure the quality of any conclusions on transcriptome diversity drawn from these data. The lack of long-read oriented quality aware analysis motivated the development of SQANTI \url{(https://bitbucket.org/ConesaLab/sqanti)}, an automated pipeline for the structural characterization and quality assessment of full-length transcriptomes. Secondly, the gene-centric nature of functional resources remained the major limitation to the extended study of functional isoform variability, especially for novel isoforms, which cannot be characterised by static databases. Thus, we designed IsoAnnot, which dynamically constructs an isoform-resolved rich database of functional annotations by using as input transcript sequences and integrating information disseminated across several databases and prediction methods. Finally, because no methods to interrogate the functional impact of PTR were available, we developed novel approaches and user-friendly tools such as tappAS \url{(http://tappas.org/)}, designed to facilitate researchers the transcriptome-wide functional study of context-specific isoform regulation. Thereby, this thesis describes the development of an analysis framework that tackles the fundamental challenges of the isoform functional analysis by providing a set of novel methods and tools that offer an unique opportunity to explore how the phenotype is specified by altering the functional characteristics of expressed isoforms. Applied to a murine neural differentiation system, our pipeline profiled the effect of isoform regulation on the inclusion of several functional elements within transcripts between motor-neuron and oligodendrocyte differentiation systems and specifically, we discovered isoform-specific transmembrane regions whose modulation by PTR might contribute to control cell type-specific mitochondrial dynamics during neural fate determination.
This work was funded by the following grants: From 2014 to 2018. FPU: Training programme for Academic Staff. Spanish Ministry of Education, FPU2013/02348. From 2016 to 2019. NOVELSEQ: Novel methods for new challenges in the analysis of high-throughput sequencing data. MINECO, BIO2015-1658-R. From 2014 to 2017. DEANN: Developing a European American NGS Network. EU Marie Curie IRSES, GA-612583.
Fuente Lorente, LDL. (2019). Development of a bioinformatics approach for the functional analysis of alternative splicing [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/124974
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Vogel, Alexander Verfasser], Björn [Akademischer Betreuer] [Usadel, Ingo Akademischer Betreuer] Kurth, and Ulrich [Akademischer Betreuer] [Schaffrath. "Long-read sequencing for de novo genome assembly in bioeconomic context / Alexander Vogel ; Björn Usadel, Ingo Kurth, Ulrich Schaffrath." Aachen : Universitätsbibliothek der RWTH Aachen, 2020. http://d-nb.info/123506946X/34.
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Zhang, Panpan. "Étude du paysage des éléments transposables sous forme d'ADN circulaire extrachromosomique et dans l'assemblage des génomes de plantes à l'aide du séquençage en lectures longues." Thesis, Université de Montpellier (2022-….), 2022. http://www.theses.fr/2022UMONG016.
Full textAbstract:
Les éléments transposables (TEs) sont des séquences d'ADN répétitives avec la capacité intrinsèque de se déplacer et de s’amplifier dans les génomes. La transposition active des TEs est liée à la formation d'ADN circulaire extrachromosomique (ADNecc). Cependant, le paysage complet de ce compartiment d’ADNecc ainsi que ces interactions avec le génome n’étaient pas bien définies. De plus, il n’existait au début de ma thèse aucun outil bioinformatique permettant d'identifier les ADNecc à partir de données de séquençage en lectures longues. Pour répondre à ces questions au cours de mon doctorat, nous avons tout d'abord développé un outil, appelé ecc_finder, pour automatiser la détection d'eccDNA à partir de séquences en lectures longues et optimisé la détection à partir de séquences de lecture courte pour caractériser la mobilité des TE. En appliquant ecc_finder aux données eccDNA-seq d'Arabidopsis, de l'homme et du blé (avec des tailles de génome allant de 120 Mb à 17 Gb), nous avons documenté l'applicabilité étendue d'ecc_finder ainsi que l’optimisation du temps de calcul, de la sensibilité et de la précision.Dans le deuxième projet, nous avons développé un outil de méta-assemblage appelé SASAR pour réconcilier les résultats de différents assemblages de génomes à partir de données de séquençage en lectures longues. Pour différentes espèces de plantes, SASAR a obtenu des assemblages de génome de haute qualité en un temps efficace et a résolu les variations structurales causées par les TE.Dans le dernier projet, nous avons utilisé le génome assemblé par SASAR et l'ADNecc détecté par ecc_finder pour caractériser les interactions entre les ADNecc et le génome. Dans les mutants épigénétiques hypométhylés d’Arabidopsis, nous avons mis en évidence le rôle de l'épigénome dans la protection de la stabilité du génome non seulement contre la mobilité des TE mais aussi envers les réarrangements génomiques et le chimérisme des gènes. Globalement, nos découvertes sur l'ADNecc, l'assemblage du génome et leurs interactions, ainsi que le développement d'outils, offrent de nouvelles perspectives pour comprendre le rôle des TE dans l'évolution adaptative des plantes à un changement rapide de l’environnementTransposable elements (TEs) are repetitive DNA sequences with the intrinsic ability to move and amplify in genomes. Active transposition of TEs is linked to the formation of extrachromosomal circular DNA (eccDNA). However, the complete landscape of this eccDNA compartment and its interactions with the genome were not well defined. In addition, at the beginning of my thesis, there were no bioinformatics tools available to identify eccDNAs from long-read sequencing data.To address these questions during my PhD, we first developed a tool, called ecc_finder, to automate eccDNA detection from long-read sequencing and optimized detection from short-read sequences to characterize TE mobility. By applying ecc_finder to Arabidopsis, human and wheat eccDNA-seq data (with genome sizes ranging from 120 Mb to 17 Gb), we documented the broad applicability of ecc_finder as well as optimization of computational time, sensitivity and accuracy.In the second project, we developed a meta-assembly tool called SASAR to reconcile the results of different genome assemblies from long-read sequencing data. For different plant species, SASAR obtained high quality genome assemblies in an efficient time and resolved structural variations caused by TEs.In the last project, we used SASAR-assembled genome and ecc_finder-detected eccDNA to characterize eccDNA-genome interactions. In Arabidopsis hypomethylated epigenetic mutants, we highlighted the role of the epigenome in protecting genome stability not only from TE mobility but also from genomic rearrangements and gene chimerism. Overall, our findings on eccDNA, genome assembly and their interactions, as well as the development of tools, offer new insights into the role of TEs in the adaptive evolution of plants to rapid environmental change
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Jaudou, Sandra. "Metadetect : detection of Shiga toxin-producing Escherichia coli with novel metagenomics approaches and its application on dairy farms in France and Germany." Electronic Thesis or Diss., Maisons-Alfort, École nationale vétérinaire d'Alfort, 2023. http://www.theses.fr/2023ENVA0004.
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Les méthodologies actuelles de caractérisation d'Escherichia coli producteur de toxine Shiga (STEC) nécessitent l'isolement de la souche, ce qui est compliqué par le fait qu'il n'existe pas de milieu d'isolement spécifique qui distingue clairement les STEC des E. coli commensaux non pathogènes. Par conséquent, obtenir des informations sur les souches en utilisant une approche métagénomique éviterait d'isoler les souches pour les caractériser complètement. Dans le cadre du projet, en collaboration avec le BfR en Allemagne, nous évaluerons si de nouvelles approches de métagénomique à lecture longue pourraient déterminer sans ambiguïté si des marqueurs spécifiques d'EHEC typiques (E. coli entérohémorragique) sont co-localisés dans une même souche. Les approches de séquençage hybrides de deuxième et troisième génération seront évaluées. Des pipelines bioinformatiques seront évalués pour analyser les résultats de l'analyse métagénomique. Ces méthodes seront appliquées dans une étude pilote pour étudier le microbiote du lait cru provenant d'exploitations laitières françaises et allemandes et pour identifier un microbiome commun associé aux STEC pathogènes. Nous essaierons de définir un système basé sur l'établissement d'un « score moléculaire » pour qualifier l'état des exploitationsCurrent methodologies for characterization of Shiga toxin-producing Escherichia coli (STEC) require strain isolation, which is complicated by the fact that there is no specific isolation medium that clearly distinguishes STECs from non-pathogenic commensal E. coli. Therefore, obtaining strain information using a metagenomics approach would avoid isolating a strain to fully characterize it. In the framework of the project, in collaboration with the BfR in Germany, we will evaluate whether new, long-read metagenomics approaches could unambiguously determine whether specific markers of typical EHECs (Enterohemorrhagic E. coli) are co-located in the same strain. Third generation hybrid sequencing approaches will be evaluated. Appropriate bioinformatic pipelines developed in collaboration with the BfR will be evaluated to analyze the metagenomic analysis results. These methods will be applied in a pilot study to study the microbiota of raw milk from French and German dairy farms and to tentatively identify a common STEC-associated microbiome. We aim to define a ‘molecular score' based system to identify the status of the farms, in line with the objective to better precise the notion of ‘STEC molecular risk assessment approach' at the farm level
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Šalanda, Vojtěch. "Optimalizace zarovnání dat z next-generation sekvenování." Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2014. http://www.nusl.cz/ntk/nusl-236077.
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This thesis presents short DNA alignment tools optimization. These short DNA reads are products of next\nobreakdash-generation sequencing technologies. The results produced by existing align\-ment tools can be influenced by various parameters. For this purpose, an optimization framework to find the optimal values of selected parameters was developed. This framework is based on differencial evolution algorithm and its main goal is to maximize the alignment accuracy. The functionality of the framework was tested on both real and generated data sets of short DNA reads. An accurate alignment is crucial for correct prediction of various genetic characteristics.
Books on the topic "Short read and long read sequencing"
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Wright, Richardson Little. The gardener's bed-book: Short and long pieces to be read in bed by those who love husbandry and green growing things of earth. New York: PAJ Publications, 1988.
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Kleiterp, Nanno. Banking for a Better World. NL Amsterdam: Amsterdam University Press, 2016. http://dx.doi.org/10.5117/9789462983519.
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When we look at all the challenges facing the world, including inequality, population migration, and climate change, we can see a role for development banking in nearly all of them. But will that role be played for good or ill? This book brings together two people who collectively draw on their forty-five years of experience in that world to argue that development banking can-and must-play a constructive role. We only need to read the news to find public outrage at tales of short-sighted greed in the financial world. But what happens when banks invest in long-term sustainability? Readers will find a fascinating example in the journey of the Dutch development bank FMO. At times global in perspective, at other moments intimately personal, Banking for a Better World interweaves candid anecdotes with development history, as well as banking lessons with client interviews, to deliver a powerful argument for a business model that generates profit through impact, and impact through profit. This is an important and accessible must-read for anyone involved in banking, business, policy making, and civil society as a whole. Banking for a Better World challenges us to start finding overlaps between our own lives and global issues and to bridge the distance between our personal needs and those of our planet.
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deRubertis, Barbara. Let's Read Together Series: Short and Long Vowel Book and Tape Packages. Kane Press, 1997.
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VERY STRANGE STORIES YOU HAVE TO READ: SHORT STORIES FROM A LONG LIFE. iUniverse.com, 2009.
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First Step Arabic: Read & Write 200+ Arabic Words with Short & Long Vowels Independently. Nisreen Neqresh Beshqoy, 2024.
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Spence, J. T., and Q. Nahar Kheya. Short Bedtime Stories From Long Ago: Tales for Children and Those who Read Them. John Spence, 2020.
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deRubertis, Barbara. Let's Read Together Series: Set 7 (5 Short Vowel, 5 Long Vowel, 5 Vowel Team Book Titles). Kane Press, 1998.
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deRubertis, Barbara. Let's Read Together Series: Set 8 (5 Short Vowel, 5 Long Vowel, 5 Vowel Team Book and Tape Titles). Kane Press, 1998.
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Bomback, Andrew. Long Days, Short Years. The MIT Press, 2022. http://dx.doi.org/10.7551/mitpress/13682.001.0001.
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How parenting became a verb, from Dr. Spock and June Cleaver to baby whispering and free-range kids. When did “parenting” become a verb? Why is it so hard to parent, and so rife with the possibility of failure? Sitcom families of the past—the Cleavers, the Bradys, the Conners—didn't seem to lose any sleep over their parenting methods. Today, parents are likely to be up late, doomscrolling on parenting websites. In Long Days, Short Years, Andrew Bomback—physician, writer, and father of three young children—looks at why it can be so much fun to be a parent but, at the same time, so frustrating and difficult to parent. It's not a “how to” book (although Bomback has read plenty of these) but a “how come” book, investigating the emergence of an immersive, all-in approach to raising children that has made parenting a competitive (and often not very enjoyable) sport. Drawing on parenting books, mommy blogs, and historical accounts of parental duties as well as novels, films, podcasts, television shows, and his own experiences as a parent, Bomback charts the cultural history of parenting as a skill to be mastered, from the laid-back Dr. Spock's 1950s childcare bible—in some years outsold only by the actual Bible—to the more rigid training schedules of Babywise. Along the way, he considers the high costs of commercialized parenting (from the babymoon on), the pressure on mothers to have it all (and do it all), scripted parenting as laid out in How to Talk So Kids Will Listen, parenting during a pandemic, and much more.
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(Introduction), Dominique Browning, ed. The Gardener's Bed-Book: Short and Long Pieces to Be Read in Bed by Those Who Love Green Growing Things (Modern Library Gardening). Modern Library, 2003.
Find full textBook chapters on the topic "Short read and long read sequencing"
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Ribeca, Paolo. "Short-Read Mapping." In Bioinformatics for High Throughput Sequencing, 107–25. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4614-0782-9_7.
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Bryant, Douglas W., and Todd C. Mockler. "De Novo Short-Read Assembly." In Bioinformatics for High Throughput Sequencing, 85–105. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4614-0782-9_6.
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Vasquez-Velez, Laura, Veera D’Mello, and Patricia Soteropoulos. "RNA Sequencing Protocols for Short-Read Sequencing." In Methods in Molecular Biology, 125–58. New York, NY: Springer US, 2024. http://dx.doi.org/10.1007/978-1-0716-4192-7_8.
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Zhou, Xueya, Suying Bao, Binbin Wang, Xuegong Zhang, and You-Qiang Song. "Short Read Mapping for Exome Sequencing." In Methods in Molecular Biology, 93–111. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-514-9_6.
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Seki, Masahide, Miho Oka, Liu Xu, Ayako Suzuki, and Yutaka Suzuki. "Transcript Identification Through Long-Read Sequencing." In Methods in Molecular Biology, 531–41. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1307-8_29.
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Bayega, Anthony, Yu Chang Wang, Spyros Oikonomopoulos, Haig Djambazian, Somayyeh Fahiminiya, and Jiannis Ragoussis. "Transcript Profiling Using Long-Read Sequencing Technologies." In Methods in Molecular Biology, 121–47. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7834-2_6.
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Gouil, Quentin. "Assembling Plant Genomes with Long-Read Sequencing." In Plant Gametogenesis, 333–42. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2253-7_22.
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Wang, Xinkun. "De Novo Genome Assembly with Long and/or Short Reads." In Next-Generation Sequencing Data Analysis, 271–91. 2nd ed. New York: CRC Press, 2023. http://dx.doi.org/10.1201/9780429329180-15.
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Philpott, Martin, Udo Oppermann, and Adam P. Cribbs. "Long-Read Single-Cell Sequencing Using scCOLOR-seq." In Methods in Molecular Biology, 259–67. New York, NY: Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-2996-3_18.
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Moraga Amador, David, Andrea B. Kohn, Yelena Bobkova, Nedka G. Panayotova, and Leonid L. Moroz. "DNA Isolation Long-Read Genomic Sequencing in Ctenophores." In Methods in Molecular Biology, 185–200. New York, NY: Springer US, 2024. http://dx.doi.org/10.1007/978-1-0716-3642-8_7.
Full textConference papers on the topic "Short read and long read sequencing"
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Iglesias, Natalia, Ignacio Garcia Labari, Flavio Spetale, Sergio Ponce, Elizabeth Tapia, and Pilar Bulacio. "Isoform Discovery in Long-Read Sequencing: Tuning Computational Pipeline." In 2024 IEEE Biennial Congress of Argentina (ARGENCON), 1–5. IEEE, 2024. http://dx.doi.org/10.1109/argencon62399.2024.10735861.
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Acher, Olivier, and Grégoire Milliez. "Camera-readable scales for motion control in automation and robotic applications." In Imaging Systems and Applications, ITh4D.2. Washington, D.C.: Optica Publishing Group, 2024. http://dx.doi.org/10.1364/isa.2024.ith4d.2.
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We present patterned position scales to measure displacements with great accuracy over short or long distances along three in-plane degrees of freedom. These scales can be read using any machine vision camera.
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Кечин, А. А., М. А. Корюков, В. С. Боробова, and М. Л. Филипенко. "FEATURES OF MUTATION DETECTION IN LONG-READ WHOLE GENOME SEQUENCING DATA." In Сборник трудов XVIII Российской конференции "РАСПРЕДЕЛЕННЫЕ ИНФОРМАЦИОННО-ВЫЧИСЛИТЕЛЬНЫЕ РЕСУРСЫ". Crossref, 2023. http://dx.doi.org/10.25743/dir.2022.16.63.019.
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Технологии секвенирования третьего поколения позволяют идентифицировать не только точечные и короткие мутации, но и протяженные перестройки. В результате генерируется огромное количество данных, которое анализируется при помощи различных методов, в том числе машинного обучения. Целью работы стала отработка подходов к выявлению различных мутаций в данных полногеномного секвенирования технологией Oxford Nanopore. В результате были подобраны оптимальные параметры для BWA и minimap2 (картирование), Pisces (точечные мутации), Sniffles и Nanovar (крупные перестройки), а также сконструированы праймеры для валидации некоторых крупных перестроек. Third-generation sequencing technologies make it possible to identify not only point and short mutations, but also large rearrangements. Therefore, a huge amount of data is generated, which is analyzed using various methods, including machine learning. The aim of the study was to develop approaches to identifying various mutations in Oxford Nanopore sequencing data. As a result, optimal parameters were selected for BWA and minimap2 (mapping), Pisces (point mutations), Sniffles and Nanovar (large rearrangements), and primers were designed to validate some large rearrangements.
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Ludlow, Andrew T., Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, and David C. Corney. "Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.sabcs18-2724.
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Ludlow, Andrew T., Mohammed E. Sayed, Aaron L. Slusher, Mark Ribick, Anisha Pancholi, Brian Sereni, Yu Qui, Elizabeth Tseng, Meredith Ashby, and David C. Corney. "Abstract 2724: A combination of short-read and long-read RNA sequencing reveals NOVA1’s role in telomere biology." In Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.am2019-2724.
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"Novel method for isoform-centered analysis of alternative splicing using a combination of long and short-read sequencing data." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Novosibirsk ICG SB RAS 2021, 2021. http://dx.doi.org/10.18699/plantgen2021-089.
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Schatz, Florian, Lars Wienbrandt, and Manfred Schimmler. "Probability Model for Boundaries of Short-Read Sequencing." In 2012 International Conference on Advances in Computing and Communications (ICACC). IEEE, 2012. http://dx.doi.org/10.1109/icacc.2012.51.
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Da Zhi Ang, Nicholas, Wei Lin Tang, Javian Zheng Han Ng, Wendy Lee, Solomonraj Wilson, Ser Mei Koh, Menaka Priyadharsani Rajapakse, et al. "159 Refining TCR clonotype identification with long-read sequencing technique." In SITC 39th Annual Meeting (SITC 2024) Abstracts, A179—A181. BMJ Publishing Group Ltd, 2024. http://dx.doi.org/10.1136/jitc-2024-sitc2024.0159.
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Mbuga, Felix, Kathy Lam, and Wendy Lee. "Machine Learning-Based Artifact Detection for Long-Read Sequencing Data." In 2023 International Conference on Computational Science and Computational Intelligence (CSCI). IEEE, 2023. http://dx.doi.org/10.1109/csci62032.2023.00103.
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Xu, Qisheng, Yong Dou, and Yanjie Sun. "Accelerating minimap2 for long-read sequencing on NUMA multi-core CPU." In CSSE 2022: 2022 5th International Conference on Computer Science and Software Engineering. New York, NY, USA: ACM, 2022. http://dx.doi.org/10.1145/3569966.3570012.
Full textReports on the topic "Short read and long read sequencing"
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Gur, Amit, Edward Buckler, Joseph Burger, Yaakov Tadmor, and Iftach Klapp. Characterization of genetic variation and yield heterosis in Cucumis melo. United States Department of Agriculture, January 2016. http://dx.doi.org/10.32747/2016.7600047.bard.
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Project objectives: 1) Characterization of variation for yield heterosis in melon using Half-Diallele (HDA) design. 2) Development and implementation of image-based yield phenotyping in melon. 3) Characterization of genetic, epigenetic and transcriptional variation across 25 founder lines and selected hybrids. The epigentic part of this objective was modified during the course of the project: instead of characterization of chromatin structure in a single melon line through genome-wide mapping of nucleosomes using MNase-seq approach, we took advantage of rapid advancements in single-molecule sequencing and shifted the focus to Nanoporelong-read sequencing of all 25 founder lines. This analysis provides invaluable information on genome-wide structural variation across our diversity 4) Integrated analyses and development of prediction models Agricultural heterosis relates to hybrids that outperform their inbred parents for yield. First generation (F1) hybrids are produced in many crop species and it is estimated that heterosis increases yield by 15-30% globally. Melon (Cucumismelo) is an economically important species of The Cucurbitaceae family and is among the most important fleshy fruits for fresh consumption Worldwide. The major goal of this project was to explore the patterns and magnitude of yield heterosis in melon and link it to whole genome sequence variation. A core subset of 25 diverse lines was selected from the Newe-Yaar melon diversity panel for whole-genome re-sequencing (WGS) and test-crosses, to produce structured half-diallele design of 300 F1 hybrids (MelHDA25). Yield variation was measured in replicated yield trials at the whole-plant and at the rootstock levels (through a common-scion grafted experiments), across the F1s and parental lines. As part of this project we also developed an algorithmic pipeline for detection and yield estimation of melons from aerial-images, towards future implementation of such high throughput, cost-effective method for remote yield evaluation in open-field melons. We found extensive, highly heritable root-derived yield variation across the diallele population that was characterized by prominent best-parent heterosis (BPH), where hybrids rootstocks outperformed their parents by 38% and 56 % under optimal irrigation and drought- stress, respectively. Through integration of the genotypic data (~4,000,000 SNPs) and yield analyses we show that root-derived hybrids yield is independent of parental genetic distance. However, we mapped novel root-derived yield QTLs through genome-wide association (GWA) analysis and a multi-QTLs model explained more than 45% of the hybrids yield variation, providing a potential route for marker-assisted hybrid rootstock breeding. Four selected hybrid rootstocks are further studied under multiple scion varieties and their validated positive effect on yield performance is now leading to ongoing evaluation of their commercial potential. On the genomic level, this project resulted in 3 layers of data: 1) whole-genome short-read Illumina sequencing (30X) of the 25 founder lines provided us with 25 genome alignments and high-density melon HapMap that is already shown to be an effective resource for QTL annotation and candidate gene analysis in melon. 2) fast advancements in long-read single-molecule sequencing allowed us to shift focus towards this technology and generate ~50X Nanoporesequencing of the 25 founders which in combination with the short-read data now enable de novo assembly of the 25 genomes that will soon lead to construction of the first melon pan-genome. 3) Transcriptomic (3' RNA-Seq) analysis of several selected hybrids and their parents provide preliminary information on differentially expressed genes that can be further used to explain the root-derived yield variation. Taken together, this project expanded our view on yield heterosis in melon with novel specific insights on root-derived yield heterosis. To our knowledge, thus far this is the largest systematic genetic analysis of rootstock effects on yield heterosis in cucurbits or any other crop plant, and our results are now translated into potential breeding applications. The genomic resources that were developed as part of this project are putting melon in the forefront of genomic research and will continue to be useful tool for the cucurbits community in years to come.
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Iselin, Columbus O'Donnell. Preliminary report on the prediction of "Afternoon Effect". Woods Hole Oceanographic Institution, December 2022. http://dx.doi.org/10.1575/1912/29562.
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With moderate or light winds and a clear sky the diurnal heating which occurs near the sea surface can cause a serious reduction in the range of submarine detection, especially on shallow targets. This has usually been called the “afternoon effect", although as will be noticed below the ranges often remain short long after sun down. The heating of surface waters which causes such sharp downward refraction can of course be noted on a bathythermograph record, provided pen vibration does not confuse the upper part of the trace. Unfortunately it is the upper 20 or 30 feet of a bathythermograph curve which in the case of ships moving faster than 12 knots is often somewhat difficult to read with sufficient certainty. Moreover, in planning a days operations it is clearly desirable to know in advance how much reduction in range may be expected from diurnal warming.
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Sherman, A., D. N. Kuhn, Y. Cohen, R. Ophir, and R. Goenaga. Exploring the polyembryonic seed trait in mango as a basis for a biotechnology platform for fruit tree crops. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2021. http://dx.doi.org/10.32747/2021.8134176.bard.
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Mango is one of the most important fruit crops. However, the biology of this fruit tree is under studied. The lack of genetic and genomic resources has limited progress in mango research and breeding. Several research groups have recently started developing genomic tools for mango by creating transcriptome and genomic data. Sexual reproduction in plants is the main pathway for the creation of new genetic combinations. In modern agriculture, breeders exploit the genetic diversity generated through sexual reproduction to develop elite cultivars; however, these cultivars require genetic stabilization before they are suitable for mass propagation for uniform crop production. In heterozygous plants such as fruit trees, vegetative propagation (cloning) is the primary path for the propagation of genetically uniform plants. Another natural plant mechanism that can create genetically uniform plants (clones) is apomixes. Apomixis is defined as asexual reproduction through seeds that lead to the production of clonal progeny whose genotype is identical to that of the mother plant. In fruit crops like citrus and mango, sporophytic apomixes result in polyembryony, where seeds contain multiple embryos, one of which is sexually originated, and the others are clones of the mother tree. As part of this research, the reference genome of mango was established as a basic platform for mango breeding and research. It was used to map two important mango traits fruit size and polyembryony. The draft genome 'Tommy Atkins' sequence was generated using NRGene de-novo Magic on high molecular weight DNA of 'Tommy Atkins,' supplemented by 10X Genomics long read sequencing to improve the initial assembly. The final 'Tommy Atkins' genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango. The availability of a genome enables the genetic dissection of important traits. We demonstrated the utility of the genome assembly and the 'Tommy Atkins' x 'Kensington Pride' map by analyzing fruit weight phenotypic data and identifying two QTLs for this trait.
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Steffenson, B. J., I. Mayrose, Gary J. Muehlbauer, and A. Sharon. ing and comparative sequence analysis of powdery mildew and leaf rust resistance gene complements in wild barley. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2021. http://dx.doi.org/10.32747/2021.8134173.bard.
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Our overall, long-term goal is to exploit the genetic diversity present in cereal wild relatives for the development of cultivars with durable disease resistance. Our specific objectives for this proposal were to: 1) Utilize Association Genetics Resistance Gene Enrichment Sequencing (AgRenSeq) to identify and clone powdery mildew and leaf rust resistance gene complements in wild barley and 2) Conduct comparative sequence analyses of the cloned resistance genes to elucidate the basis of their specificity and evolution. The deployment of resistant cultivars is the most effective, economically efficient, and environmentally sound means of controlling plant diseases, especially in small grain cereals. The systems selected for study in this proposal are barley (Hordeum vulgare ssp. vulgare, Hvv), its wild progenitor (Hordeum vulgare ssp. spontaneum, Hvs) and the powdery mildew (Blumeria graminis f. sp. hordei, Bgs) and leaf rust (Puccinia hordei, Ph) pathogens. We compiled a diverse panel of Hvs accessions (the Wild Barley Diversity Collection or WBDC; N = 314) from across its native range and evaluated it to 40 isolates of Bgs and 12 isolates of Ph. We obtained genomic DNA sequences enriched for Nucleotide Binding Site-Leucine Rich Repeat (NLR) type resistance genes for 203 WBDC accessions, plus cultivar Morex for which the first reference genome sequence of barley was based. We assembled the 250 bp Illumina sequencing reads into contigs using CLC assembly cell. From this effort, we successfully assembled the sequences of 201 WBDC accessions plus Morex and used NLR Parser to identify contigs containing NLR genes. AgRenSeq was then used to identify k-mers (short oligonucleotide sequences of length k) that were associated with resistance to each isolate of the two pathogens. This analysis was performed individually for all WBDC accessions and each individual pathogen race (9,898 host accession x pathogen race combinations). We visualized the results from these analyses in Manhattan plots and identified 311 and 144 peaks for powdery mildew and leaf rust resistance, respectively. The next step in the analysis was to identify the contigs associated with the peaks in the Manhattan plots. BLAST (Basic Local Alignment Search Tool) searches were employed to identify closely related contigs in other WBDC accessions or in Morex. We identified two candidate R genes that were only present in resistant WBDC accessions. One of these was present in seven WBDC lines and was associated with resistance to four leaf rust isolates. BLAST analysis of this gene revealed that it was Rph15, one of the most widely effective leaf rust resistance genes reported in Hordeum. This gene was cloned and functionally validated in association with our Australian colleagues (Cheng et al., 2021). We are currently in the process of cloning six of other resistance genes: four for powdery mildew and two for leaf rust. As the contigs do not contain much of the promoter sequences, we have employed a genome walking approach to identify 2,500 bp of promoter sequence. To speed up and simplify the cloning of resistance genes from the WBDC, the PI established the International Wild Barley Sequencing Consortium (IWBSC; https://iwbsc.umn.edu/) comprised of over 60 researchers from 14 different countries and raised over $150,000 through crowdfunding to pay for 10X depth sequence coverage. Genome-wide association study (GWAS) of whole genome sequencing (WGS) data identified extremely strong and clear signals of association for several resistance genes which will facilitate gene cloning in concert with a wild barley pan-genome currently under construction. The cloning of multiple resistance gene can facilitate the development of durably resistant cultivars by inserting, through transgenesis, cassettes of multiple resistance genes.
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Azzi, Elias S., Cecilia Sundberg, Helena Söderqvist, Tom Källgren, Harald Cederlund, and Haichao Li. Guidelines for estimation of biochar durability : Background report. Department of Energy and Technology, Swedish University of Agricultural Sciences, 2023. http://dx.doi.org/10.54612/a.lkbuavb9qc.
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Biochar is produced by heating biomass in the total or partial absence of oxygen. This report addresses the long-term persistence of biochar in soil and how this can be managed in climate calculations and reporting. The report consists of this summary and four chapters, which can be read independently. Different terms have been used to describe the durability of biochar carbon storage, but also the physical presence of biochar in soils, e.g. persistence, permanence, recalcitrance, residence times, stability. Today, the term “durability of carbon storage” is preferred in policy contexts, but various academic disciplines such as soil science have other established terms like “persistence”. Here, both durability and persistence are used, rather interchangeably. It is important to be aware of differences in meaning that exist between disciplines. The purpose of this report is to present the state of knowledge regarding the proportion of carbon in biochar that remains in the soil over time and provide recommendations for calculating this. There is a need to calculate the persistence of biochar in soil for national climate reporting, corporate climate reporting, carbon credit trading, and life cycle assessments for various purposes. On the persistence of biochar The amount of biochar remaining after a certain time depends on the properties of the biochar and the environment in which it is located. Nearly all research on biochar persistence has focused on its application in agricultural soils. The main reason for the high durability of biochar carbon storage is the formation of fused aromatic stable structures during biomass pyrolysis. A high degree of fused aromatic structures makes biochar much less prone to microbial decomposition than fresh biomass. Different biochars have different properties, and this influences how long they persist in the soil. To achieve biochar with properties that provide higher persistence, it should be produced at higher temperatures for a sufficient duration. Measuring and calculating biochar persistence Established quantification methods of 100-year biochar persistence (e.g. referenced in IPCC inventory guidelines and used in voluntary carbon markets, to date) extrapolate short-term soil decomposition processes, and do not fully consider the processes that may explain millennial persistence. Calculations regarding biochar persistence have traditionally used a time span of 100 years to describe the amount of remaining carbon after a certain time. The use of specifically 100 years lacks a well founded scientific reason, but has been regarded as “far enough” into the future from a climate perspective and close enough for modelling to be meaningful. An active area of research relevant for the understanding biochar carbon storage durability is the development of advanced analytical characterisation methods of biochar that will enable measurement of the physicochemical heterogeneity in carbon structures present in biochar. Another area of continued research is biochar incubation, with a focus on field conditions, to elucidate both differences from laboratory conditions, and how transport processes affect biochar in the field. Recommendation and conclusion In the project, available research data has been aggregated into a functional model that calculates how much of the carbon in biochar remains after a given number of years. The model is based on the H/C ratio of the biochar placed in the soil and the annual average temperature at the location. The model is made freely accessible to provide biochar market actors with the best available knowledge for estimating the durability of biochar carbon. Existing research results provide a sufficient foundation for estimation of the amount of biochar expected to remain over time. Future research results are expected to lead to increased knowledge regarding the decomposition properties of biochar, in particular biochars with a very low H/C ratio. Therefore, this recommendation will be revised by the end of the project in 2025.