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Relevant bibliographies by topics / Single cell mRNA sequencing

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Academic literature on the topic 'Single cell mRNA sequencing'

Author: Grafiati

Published: 25 January 2025

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Journal articles on the topic "Single cell mRNA sequencing"

1

Xu, Yunbo, Hongliang Hu, Jie Zheng, and Biaoru Li. "Feasibility of Whole RNA Sequencing from Single-Cell mRNA Amplification." Genetics Research International 2013 (December 23, 2013): 1–8. http://dx.doi.org/10.1155/2013/724124.

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Single-cell sampling with RNA-seq analysis plays an important role in reference laboratory; cytogenomic diagnosis for specimens on glass-slides or rare cells in circulating blood for tumor and genetic diseases; measurement of sensitivity and specificity in tumor-tissue genomic analysis with mixed-cells; mechanism analysis of differentiation and proliferation of cancer stem cell for academic purpose. Our single- cell RNA-seq technique shows that fragments were 250–450 bp after fragmentation, amplification, and adapter addition. There were 11.6 million reads mapped in raw sequencing reads (19.6 million). The numbers of mapped genes, mapped transcripts, and mapped exons were 31,332, 41,210, and 85,786, respectively. All QC results demonstrated that RNA-seq techniques could be used for single-cell genomic performance. Analysis of the mapped genes showed that the number of genes mapped by RNA-seq (6767 genes) was much higher than that of differential display (288 libraries) among similar specimens which we have developed and published. The single-cell RNA-seq can detect gene splicing using different subtype TGF-beta analysis. The results from using Q-rtPCR tests demonstrated that sensitivity is 76% and specificity is 55% from single-cell RNA-seq technique with some gene expression missing (2/8 genes). However, it will be feasible to use RNA-seq techniques to contribute to genomic medicine at single-cell level.

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Dhar, Manjima, Reem Khojah, Andy Tay, and Dino Di Carlo. "Research highlights: microfluidic-enabled single-cell epigenetics." Lab on a Chip 15, no. 21 (2015): 4109–13. http://dx.doi.org/10.1039/c5lc90101d.

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Barcoded droplet-based mRNA sequencing from thousands of single cells – Exploring regulatory differences in single cells with fluidics-controlled ATAC-seq – Towards single-cell chromatin immunoprecipitation.

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D’avola, D., C. Villacorta, S. N. Martins-Filho, A. Craig, I. Labgaa, J. V. Felden, A. Kimaada, et al. "Single-cell mRNA sequencing to characterize circulating tumor cells in hepatocellular carcinoma." Journal of Hepatology 68 (April 2018): S445—S446. http://dx.doi.org/10.1016/s0168-8278(18)31131-0.

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Nakamoto, Margaret, Mirko Corselli, Ian Taylor, and Suraj Saksena. "Single cell multiomic analysis of chronically stimulated T cells displaying hallmarks of T-cell exhaustion." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 189.18. http://dx.doi.org/10.4049/jimmunol.202.supp.189.18.

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Abstract T-cell exhaustion is a dysfunctional state caused by persistent antigen stimulation mediated by chronic infections or cancer. Current immunotherapy approaches focus on reinvigorating exhausted T cells through immune-receptor-blocking antibodies. A deeper understanding of the exhausted T-cell state is crucial for identification of new therapeutic targets. Single cell RNA-sequencing (scRNA-seq) is a powerful tool for resolving heterogeneity in complex systems like these; however the lack of protein information for the same cells can make it difficult to identify cells of interest or detect cellular responses. To resolve both mRNA and protein information in the same assay, we utilized DNA-conjugated BD™ AbSeq antibodies to profile protein and mRNA expression, using high-throughput sequencing analysis at the single cell level. Sorted T cells that had been transiently and chronically stimulated were multiplexed using the BD™ Single-Cell Multiplexing Kit and pooled before undergoing cell partitioning and cDNA capture on the BD Rhapsody™ system; this minimized batch effects and saved time and reagents. A panel of 400 mRNA targets alongside the 39-plex antibody panel was used for the study. We showed that cell-type resolution increased with higher protein plex, and the addition of mRNA information alongside protein markers revealed candidate protein markers for specific cell states. This study showcased the power of multiomics in analyzing heterogenous cell populations and cell states and answering complex biological questions. Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company. © 2019 BD and its subsidiaries. All rights reserved.

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Lee, Jeongwoo, Do Young Hyeon, and Daehee Hwang. "Single-cell multiomics: technologies and data analysis methods." Experimental & Molecular Medicine 52, no. 9 (September 2020): 1428–42. http://dx.doi.org/10.1038/s12276-020-0420-2.

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Abstract Advances in single-cell isolation and barcoding technologies offer unprecedented opportunities to profile DNA, mRNA, and proteins at a single-cell resolution. Recently, bulk multiomics analyses, such as multidimensional genomic and proteogenomic analyses, have proven beneficial for obtaining a comprehensive understanding of cellular events. This benefit has facilitated the development of single-cell multiomics analysis, which enables cell type-specific gene regulation to be examined. The cardinal features of single-cell multiomics analysis include (1) technologies for single-cell isolation, barcoding, and sequencing to measure multiple types of molecules from individual cells and (2) the integrative analysis of molecules to characterize cell types and their functions regarding pathophysiological processes based on molecular signatures. Here, we summarize the technologies for single-cell multiomics analyses (mRNA-genome, mRNA-DNA methylation, mRNA-chromatin accessibility, and mRNA-protein) as well as the methods for the integrative analysis of single-cell multiomics data.

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Shi, Xiaoshan, Imteaz Siddique, Margaret Nakamoto, and Stefanie Mortimer. "Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 246.17. http://dx.doi.org/10.4049/jimmunol.204.supp.246.17.

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Abstract High-throughput single cell RNA-seq (scRNA-seq) has transformed our understanding of complex and heterogenous immune populations. New advances in scRNA-seq are expanding the molecules that can be profiled at the single cell level, such as oligo-conjugated antibody technologies that enable protein expression profiling alongside mRNA. Although the ability to sequence the immune repertoire can provide crucial insights into understanding the complexities of the adaptive immune system and advancing discoveries in immuno-oncology, the ability to extract this information from single cells requires new technology to profile regions of the mRNA that are missed by conventional 3′ scRNA-seq. In this study we utilized an ex vivo antigen stimulation system to measure antigen-specific T-cell activation and clonal amplification. Stimulated T cells from two donors were loaded onto the BD Rhapsody™ Single-Cell Analysis System to extract immune repertoire information in addition to gene and protein expression information from the same cells. In a single workflow, we profiled a panel of 400 mRNA targets, 20 BD® AbSeq protein markers associated with different status of T-cell activation, and the hypervariable region of T-cell receptors at the single cell resolution. The study is a proof of concept of combining mRNA, protein and immune repertoire analysis at the single cell level to deconvolute the heterogeneity and different activation of stimulated T cells. For Research Use Only. Not for use in diagnostic or therapeutic procedures. BD, the BD Logo, and Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2019 BD. All rights reserved.

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Liu, Wendao, and Noam Shomron. "Analysis of MicroRNA Regulation and Gene Expression Variability in Single Cell Data." Journal of Personalized Medicine 12, no. 10 (October 21, 2022): 1750. http://dx.doi.org/10.3390/jpm12101750.

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MicroRNAs (miRNAs) regulate gene expression by binding to mRNAs, and thus reduce target gene expression levels and expression variability, also known as ‘noise’. Single-cell RNA sequencing (scRNA-seq) technology has been used to study miRNA and mRNA expression in single cells. To evaluate scRNA-seq as a tool for investigating miRNA regulation, we analyzed datasets with both mRNA and miRNA expression in single-cell format. We found that miRNAs slightly reduce the expression noise of target genes; however, this effect is easily masked by strong technical noise from scRNA-seq. We suggest improvements aimed at reducing technical noise, which can be implemented in experimental design and computational analysis prior to running scRNA-seq. Our study provides useful guidelines for experiments that evaluate the effect of miRNAs on mRNA expression from scRNA-seq.

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Schoettle, Louis, Xixi Wei, Marlene Garcia-Neurer, Veronkia Zarnitsyna, Rustom Antia, Hao Yan, and Joseph Blattman. "DNA origami: single cell analysis of T cell receptors without single cell sorting. (LYM7P.719)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 193.7. http://dx.doi.org/10.4049/jimmunol.192.supp.193.7.

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Abstract The immune system must be able to recognize virtually any pathogen (diversity) while maintaining enough cells specific for each pathogen in order to mount an effective response (protection). T cells generate diversity by imprecise joining of gene segments to generate alpha/beta heterodimeric receptors. Linking sequence information for TCRα and TCRβ from individual cells has been problematic due to the cost of single cell sorting and inadequate molecular approaches for linking mRNA encoding these proteins. We have developed novel DNA origami nanostructures to capture and protect both TCRα and TCRβ mRNA from individual cells, which can then be physically linked via a unique dual-primed reverse-transcription and ligation reaction to generate single amplicons containing both TCR from individual cells for use in next generation sequencing. We have demonstrated high efficiency transfection and recovery of DNA origami, optimized methods for purification with bound TCR mRNA, and validated this approach with transgenic T cells expressing a known TCR sequence. Surprisingly, we find that <1% of TCR are shared even between genetically identical individuals, challenging the idea of “public” TCR. This approach is directly amenable to single cell analysis of other immune receptors (or other species) by relatively simple modifications of the origami sequences, and could be applied to virtually any heterogeneous cell population for which sequence information on any two genes is required.

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Li, Jiawei, Yi Zhang, Cheng Yang, and Ruiming Rong. "Discrepant mRNA and Protein Expression in Immune Cells." Current Genomics 21, no. 8 (December 21, 2020): 560–63. http://dx.doi.org/10.2174/1389202921999200716103758.

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With the development of single-cell mRNA sequencing (scRNA-seq), researchers have attempted to identify new methods for performing in-depth studies of immune cells. However, the discrepancies between the mRNA levels and the levels of surface proteins have confused many researchers. Here, we report a significant and interesting phenomenon in which the mRNA and protein expression levels were mismatched in immune cells. We concluded that scRNA-seq should be combined with other sequencing methods in single-cell studies (e.g., CITE-seq). The simultaneous assessment of both mRNA and protein expression will enhance the precision and credibility of the results.

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Battich, Nico, Joep Beumer, Buys de Barbanson, Lenno Krenning, Chloé S. Baron, Marvin E. Tanenbaum, Hans Clevers, and Alexander van Oudenaarden. "Sequencing metabolically labeled transcripts in single cells reveals mRNA turnover strategies." Science 367, no. 6482 (March 5, 2020): 1151–56. http://dx.doi.org/10.1126/science.aax3072.

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The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.

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Dissertations / Theses on the topic "Single cell mRNA sequencing"

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Johnson, Travis Steele. "Integrative approaches to single cell RNA sequencing analysis." The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1586960661272666.

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Borgström, Erik. "Technologies for Single Cell Genome Analysis." Doctoral thesis, KTH, Genteknologi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-181059.

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During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools.

QC 20160127

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La, Forest Divonne Sébastien De. "Caractérisation constitutive et en condition d'infection bactérienne des populations hémocytaires par une approche intégrative cytologiques, transcriptomiques et fonctionnelles chez l'huitre creuse Crassostrea (Magallana) gigas." Electronic Thesis or Diss., Perpignan, 2024. http://www.theses.fr/2024PERP0025.

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L'huître du Pacifique, Crassostrea (Magallana) gigas, est un mollusque bivalve d'une importance écologique et économique majeure, et constitue désormais une espèce modèle émergente dans l'étude de l'immunité innée des bivalves. Ces dernières années, les élevages d'huîtres ont été confrontés à des épisodes de mortalités massives, exacerbés par le changement climatique et les activités anthropiques. Ces mortalités, bien que multifactorielles, partagent un point commun : la capacité des pathogènes (virus et bactéries) à échapper aux défenses immunitaires des huîtres, entraînant des septicémies souvent fatales. Alors que chez les vertébrés, et particulièrement l'Homme, les cellules immunitaires sont bien caractérisées, la diversité et la spécialisation fonctionnelle des hémocytes chez C. gigas restent encore une boite noire apprement débattue au sein de la communauté scientifique. Ce manque de connaissances entrave la compréhension des interactions hôte-pathogènes, limitant ainsi le développement de stratégies pour réduire les mortalités en ostréiculture. Dans ce contexte, l'objectif principal de mon projet de thèse a été de caractériser les types hémocytaires circulants de C. gigas, en utilisant des approches cytologiques, fonctionnelles et de transcriptomique sur cellules uniques (scRNA-seq). Ces méthodes ont permis dans un premier temps d'identifier sept types hémocytaires distincts constitutifs chez les animaux naifs. Ces populations hémocytaires ont été caractérisées par leurs propriétés morphologiques, leurs profils d'expression génique et leurs fonctions biologiques spécifiques. De plus, nous avons établi une ontologie des hémocytes, suggérant des voies potentielles de différenciation des lignages cellulaires. Grâce à cet atlas hémocytaire, nous avons alors pu évaluer l'impact différentiel d'une infection par Vibrio aestuarianus sur les populations hémocytaires, à la fois du point de vue cytologique et transcriptomique, révélant des altérations dépendantes de la charge bactérienne circulante. Ces travaux apportent une contribution significative à la compréhension de l'immunité chez C. gigas, en permettant une définition précise des types hémocytaires. Nos résultats proposent un atlas hémocytaire de référence et soulignent l'importance de l'étude de l'homéostasie hémocytaire des mollusques pour mieux comprendre et anticiper les crises ostréicoles lors d'épisodes de mortalité
The Pacific oyster, Crassostrea (Magallana) gigas, is a bivalve mollusk of significant ecological and economic importance, and it has recently emerged as a model species for studying the innate immunity of bivalves. In recent years, oyster farming has been confronted with episodes of massive mortality, exacerbated by climate change and human activities. These mortalities, though multifactorial, share a common factor: the ability of pathogens (viruses and bacteria) to evade the oysters' immune defenses, often leading to fatal septicemia. While immune cells in vertebrates, particularly humans, are well characterized, the diversity and functional specialization of hemocytes in C. gigas remain a black box and are hotly debated within the scientific community. This knowledge gap hampers our understanding of host-pathogen interactions, thus limiting the development of strategies to reduce oyster mortality in aquaculture. In this context, the main objective of my thesis project was to characterize the circulating hemocyte types in C. gigas using cytological, functional, and single-cell transcriptomic approaches (scRNA-seq). These methods first allowed us to identify seven distinct constitutive hemocyte types in naïve animals. These hemocyte populations were characterized based on their morphological properties, gene expression profiles, and specific biological functions. Furthermore, we established a hemocyte ontology, suggesting potential differentiation pathways for the cell lineages. Using this hemocyte atlas, we then assessed the differential impact of Vibrio aestuarianus infection on hemocyte populations, both from a cytological and transcriptomic perspective, revealing alterations dependent on the circulating bacterial load. This work provides a significant contribution to the understanding of immunity in C. gigas, by offering a precise definition of hemocyte types. Our results propose a reference hemocyte atlas and emphasize the importance of studying hemocyte homeostasis in mollusks to better understand and anticipate oyster mortality crises during epizootic episodes

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Raoux, Corentin. "Review and Analysis of single-cell RNA sequencing cell-type identification and annotation tools." Thesis, KTH, Skolan för kemi, bioteknologi och hälsa (CBH), 2021. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-297852.

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Single-cell RNA-sequencing makes possible to study the gene expression at the level of individual cells. However, one of the main challenges of the single-cell RNA-sequencing analysis today, is the identification and annotation of cell types. The current method consists in manually checking the expression of genes using top differentially expressed genes and comparing them with related cell-type markers available in scientific publications. It is therefore time-consuming and labour intensive. Nevertheless, in the last two years,numerous automatic cell-type identification and annotation tools which use different strategies have been created. But, the lack of specific comparisons of those tools in the literature and especially for immuno-oncologic and oncologic purposes makes difficult for laboratories and companies to know objectively what are the best tools for annotating cell types. In this project, a review of the current tools and an evaluation of R tools were carried out.The annotation performance, the computation time and the ease of use were assessed. After this preliminary results, the best selected R tools seem to be ClustifyR (fast and rather precise) and SingleR (precise) for the correlation-based tools, and SingleCellNet (precise and rather fast) and scPred (precise but a lot of cell types remains unassigned) for the supervised classificationtools. Finally, for the marker-based tools, MAESTRO and SCINA are rather robust if they are provided with high quality markers.

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Kindblom, Marie, and Hakim Ezeddin Al. "Phylogenetic fatemapping: estimating allelic dropout probability in single cell genomic sequencing." Thesis, KTH, Skolan för datavetenskap och kommunikation (CSC), 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-186453.

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Single-cell genomic sequencing is a rapidly developing field that will play a vital role in human biology and science in the future. As of now, next-generation sequencing is accelerating in speed and decreasing in cost more quickly than Moore's law. Studies have shown that all cells in the human body have with very high probability a unique genomic signature, due to the somatic evolution which have accumulated mutations starting from the zygotic state. The possible reconstruction of phylogenetic lineage trees would be of vital importance to several fields in medicine, such as the stem cell research field. However, state-of-the-art methods for amplification such as WGA currently suffers from extensive allelic dropout which is troublesome when reconstructing phylogenetic trees. We have constructed a statistical model that can be used to predict site specific allelic dropout. Our results suggests that logistic regression is a suitable method for modelling allelic dropout, and that there is a non-linear relationship between the read depth and distance.

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Henao, Diaz Emanuela. "Towards single-cell exome sequencing with spatial resolution in tissue sections." Thesis, KTH, Skolan för bioteknologi (BIO), 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-150564.

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Evrony, Gilad David. "Single-cell Sequencing Studies of Somatic Mutation in the Human Brain." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10747.

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A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurologic disease. To address this question, we developed methods to amplify genomes of single neurons from human brains, achieving >80% genome coverage of single-cells and allowing study of a wide-range of somatic mutation types.

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Ke, Rongqin. "Detection and Sequencing of Amplified Single Molecules." Doctoral thesis, Uppsala universitet, Molekylära verktyg, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-183141.

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Improved analytical methods provide new opportunities for both biological research and medical applications. This thesis describes several novel molecular techniques for nucleic acid and protein analysis based on detection or sequencing of amplified single molecules (ASMs). ASMs were generated from padlock probe assay and proximity ligation assay (PLA) through a series of molecular processes. In Paper I, a simple colorimetric readout strategy for detection of ASMs generated from padlock probe assay was used for highly sensitive detection of RNA virus, showing the potential of using padlock probes in the point-of-care diagnostics. In Paper II, digital quantification of ASMs, which were generated from padlock probe assay and PLA through circle-to-circle amplification (C2CA), was used for rapid and sensitive detection of nucleic acids and proteins, aiming for applications in biodefense. In Paper III, digital quantification of ASMs that were generated from PLA without C2CA was shown to be able to improve the precision and sensitivity of PLA when compared to the conventional real-time PCR readout. In Paper IV, a non-optical approach for detection of ASMs generated from PLA was used for sensitive detection of bacterial spores. ASMs were detected through sensing oligonucleotide-functionalized magnetic nanobeads that were trapped within them. Finally, based on in situ sequencing of ASMs generated via padlock probe assay, a novel method that enabled sequencing of individual mRNA molecules in their natural context was established and presented in Paper V. Highly multiplex detection of mRNA molecules was also achieved based on in situ sequencing. In situ sequencing allows studies of mRNA molecules from different aspects that cannot be accessed by current in situ hybridization techniques, providing possibilities for discovery of new information from the complexity of transcriptome. Therefore, it has a great potential to become a useful tool for gene expression research and disease diagnostics.

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Tu, Ang A. (Ang Andy). "Recovery of T cell receptor variable sequences from 3' barcoded single-cell RNA sequencing libraries." Thesis, Massachusetts Institute of Technology, 2020. https://hdl.handle.net/1721.1/127888.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, May, 2020
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 107-112).
Heterogeneity of the immune system has increasingly necessitated the use of high-resolution techniques, including flow cytometry, RNA-seq, and mass spectrometry, to decipher the immune underpinnings of various diseases such as cancer and autoimmune disorders. In recent years, high-throughput single-cell RNA sequencing (scRNA-seq) has gained popularity among immunologists due to its ability to effectively characterize thousands of individual immune cells from tissues. Current techniques, however, are limited in their ability to elucidate essential immune cell features, including variable sequences of T cell antigen receptors (TCRs) that confer antigen specificity. Incorporation of TCR sequencing into scRNA-seq data could identify cells with shared antigen-recognition, further elucidating dynamics of antigen-specific immune responses in T cells.
In the first part of this thesis work, we develop a strategy that enables simultaneous analysis of TCR sequences and corresponding full transcriptomes from 32 barcoded scRNA-seq samples. This approach is compatible with common 32 scRNA-seq methods, and adaptable to processed samples post hoc. We applied the technique to identify transcriptional signatures associated with clonal T cells from murine and human samples. In both cases, we observed preferential phenotypes among subsets of expanded T cell clones, including cytotoxic T cell states associated with immunization against viral peptides. In the second part of the thesis, we apply the strategy to a 12-patient study of peanut food allergy to characterize T helper cell responses to oral immunotherapy (OIT). We identified clonal T cells associated with distinct subsets of T helper cells, including Teff, Treg, and Tfh, as well as Th1, Th2, and Th17 signatures.
We found that though the TCR repertoires of the patients were remarkably stable, regardless of their clinical outcomes, Th1 and Th2 clonotypes were phenotypically suppressed while Tfh clonotypes were not affected by therapy. Furthermore, we observed that highly activated clones were less likely to be suppressed by OIT than less activated clones. Our work represents one of the most detailed transcriptomic profiles of T helper cells in food allergy. In the last part of the thesis, we leverage the simplicity and adaptability of the method to recover TCR sequences from previously processed scRNA-seq samples derived from HIV patients and a nonhuman primate model of TB. In the HIV study, we recovered expanded clonotypes associated with activated T cells from longitudinal samples from patients with acute HIV infections. In the TB study, we modified the primers used in the method to T cells from TB granulomas of cynomolgus macaques.
We identified not only expanded clonotypes associated with cytotoxic functions, but also clonotypes shared by clusters of activated T cells. In total, these results demonstrate the utility of our method when studying diseases in which clonotype-driven responses are critical to understanding the underlying biology.
by Ang A. Tu.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

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Lefebvre, Keely. "Resolving the Taxonomy and Phylogenetics of Benthic Diatoms from Single Cell Sequencing." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/34553.

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Benthic diatoms are often used as indicators of water quality and past environmental conditions. This depends entirely on a reliable taxonomic system. With the advent of DNA techniques, genetic analyses can now be used in tandem with traditional microscopy in order to improve taxonomy and determine evolutionary relationships. This thesis examined a speciose genus of diatoms Neidium (> 300 species) and, using sequence data from molecular markers as well as traditional morphological analyses, investigated phylogenetic relationships. Fresh benthic samples from aquatic ecosystems in Eastern North America were collected; Neidium taxa were examined using light and scanning electron microscopy then compared to the original specimen types. A total of 124 individual cells were retrieved, amplified, and sequenced for four molecular markers (rbcL, 18S, psbA, and psbC). Phylogenetic reconstructions were completed using Maximum likelihood and Bayesian analyses; when compared with morphological analyses this led to the delineation of several novel Neidium species.

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Books on the topic "Single cell mRNA sequencing"

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Suzuki, Yutaka, ed. Single Molecule and Single Cell Sequencing. Singapore: Springer Singapore, 2019. http://dx.doi.org/10.1007/978-981-13-6037-4.

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Yu, Buwei, Jiaqiang Zhang, Yiming Zeng, Li Li, and Xiangdong Wang, eds. Single-cell Sequencing and Methylation. Singapore: Springer Singapore, 2020. http://dx.doi.org/10.1007/978-981-15-4494-1.

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Wang, Xiangdong, ed. Single Cell Sequencing and Systems Immunology. Dordrecht: Springer Netherlands, 2015. http://dx.doi.org/10.1007/978-94-017-9753-5.

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Ding, Hongxu. Understand Biology Using Single Cell RNA-Sequencing. [New York, N.Y.?]: [publisher not identified], 2018.

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Levitin, Hanna M. Biological Inference from Single Cell RNA-Sequencing. [New York, N.Y.?]: [publisher not identified], 2020.

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Suzuki, Yutaka. Single Molecule and Single Cell Sequencing. Springer, 2019.

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Menon, Swapna. Single Cell Sequencing Essentials in Brief: Single Cell RNA Sequencing and Orthogonal Omics Technologies. Independently Published, 2021.

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Wang, Xiangdong. Single Cell Sequencing and Systems Immunology. Springer, 2015.

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Wang, Xiangdong. Single Cell Sequencing and Systems Immunology. Springer, 2016.

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Proserpio, Valentina. Single Cell Methods: Sequencing and Proteomics. Springer New York, 2019.

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Book chapters on the topic "Single cell mRNA sequencing"

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Livak, Kenneth J. "Eukaryotic Single-Cell mRNA Sequencing." In Field Guidelines for Genetic Experimental Designs in High-Throughput Sequencing, 343–65. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-31350-4_14.

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Shum, Eleen Y., Elisabeth M. Walczak, Christina Chang, and H. Christina Fan. "Quantitation of mRNA Transcripts and Proteins Using the BD Rhapsody™ Single-Cell Analysis System." In Single Molecule and Single Cell Sequencing, 63–79. Singapore: Springer Singapore, 2019. http://dx.doi.org/10.1007/978-981-13-6037-4_5.

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Vanlandewijck, Michael, and Christer Betsholtz. "Single-Cell mRNA Sequencing of the Mouse Brain Vasculature." In Methods in Molecular Biology, 309–24. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8712-2_21.

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De Simone, Marco, Grazisa Rossetti, and Massimiliano Pagani. "Chromium 10× Single-Cell 3′ mRNA Sequencing of Tumor-Infiltrating Lymphocytes." In Methods in Molecular Biology, 87–110. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9240-9_7.

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Meyer, Michelle, and Palaniappan Ramanathan. "Droplet-Based Single-Cell 3′ mRNA Sequencing of Marburg Virus-Infected Samples." In Methods in Molecular Biology, 387–405. New York, NY: Springer US, 2024. http://dx.doi.org/10.1007/978-1-0716-4256-6_27.

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Sanada, Chad D., and Aik T. Ooi. "Single-Cell Dosing and mRNA Sequencing of Suspension and Adherent Cells Using the PolarisTM System." In Methods in Molecular Biology, 185–95. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9240-9_12.

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Durruthy-Durruthy, Robert, and Manisha Ray. "Using Fluidigm C1 to Generate Single-Cell Full-Length cDNA Libraries for mRNA Sequencing." In Methods in Molecular Biology, 199–221. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7471-9_11.

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Barreby, Emelie, and Connie Xu. "Kupffer Cell mRNA Sequencing." In Methods in Molecular Biology, 27–44. New York, NY: Springer US, 2020. http://dx.doi.org/10.1007/978-1-0716-0704-6_5.

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Zhu, Chenxu, Yun Gao, Jinying Peng, Fuchou Tang, and Chengqi Yi. "Single-Cell 5fC Sequencing." In Methods in Molecular Biology, 251–67. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9240-9_16.

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Kohn, Andrea B., Tatiana P. Moroz, Jeffrey P. Barnes, Mandy Netherton, and Leonid L. Moroz. "Single-Cell Semiconductor Sequencing." In Methods in Molecular Biology, 247–84. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-556-9_18.

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Conference papers on the topic "Single cell mRNA sequencing"

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Barmpas, Petros, Sotiris K. Tasoulis, Spiros V. Georaakonoulos, and Vassilis P. Plagianakos. "Hyperdimensional Computing Approaches in Single Cell RNA Sequencing Classification." In 2024 IEEE Congress on Evolutionary Computation (CEC), 1–8. IEEE, 2024. http://dx.doi.org/10.1109/cec60901.2024.10612207.

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Hu, Can, and Yuxia Sheng. "Hypergraph-based Clustering for Single-Cell RNA Sequencing Data." In 2024 43rd Chinese Control Conference (CCC), 3529–34. IEEE, 2024. http://dx.doi.org/10.23919/ccc63176.2024.10662510.

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Dong, Bowen, Sarabjot Pabla, Vincent Giamo, Weijian Jiang, Kiyomi Taniguchi, Sean T. Glenn, Masataka Shirai, and Pawel Kalinski. "Abstract 968: Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-968.

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Siddique, Imteaz, Xiaoshan Shi, Margaret Nakamoto, and Stefanie Mortimer. "Abstract 1344: Simultaneous mRNA, protein, and immune repertoire profiling of antigen-specific T cells by single cell sequencing." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-1344.

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Trump, S., J. Loske, M. T. Völker, O. Debnath, S. Lukassen, N. Ishaque, M. Messingschlager, et al. "Single cell mRNA sequencing of nasal swabs indicate an impaired ciliated cell function in COVID-19 convalescents with persisting dyspnea." In ERS International Congress 2022 abstracts. European Respiratory Society, 2022. http://dx.doi.org/10.1183/13993003.congress-2022.4377.

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Egidio, Camila, Michael Gonzales, Joel Brockman, Shuwen Chen, Robert Durruthy-Durruthy, Clare Rogers, and Manisha Ray. "Abstract LB-078: Improved single-cell mRNA sequencing for transcriptome and paired-chain TCR analysis of primary human CD3+ T cells." In Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-lb-078.

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Seinstra, D., L. Kester, D. Van der Velden, J. Wesseling, E. Voest, A. Van Oudenaarden, and J. Van Rheenen. "PO-337 Single cell mRNA sequencing reveals the presence of the gene expression signature of all major molecular subtypes in individual breast cancers." In Abstracts of the 25th Biennial Congress of the European Association for Cancer Research, Amsterdam, The Netherlands, 30 June – 3 July 2018. BMJ Publishing Group Ltd, 2018. http://dx.doi.org/10.1136/esmoopen-2018-eacr25.849.

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Giamo, Vincent, Melissa Grimm, Sarabjot Pabla, Ellen Karasik, Jesse Luce, Sean T. Glenn, Jeffrey Conroy, et al. "Abstract 970: Single-cell mRNA sequencing analysis of the synergistic impact of double-stranded RNA (dsRNA) and IFNα on human monocyte-derived macrophages." In Proceedings: AACR Annual Meeting 2020; April 27-28, 2020 and June 22-24, 2020; Philadelphia, PA. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1538-7445.am2020-970.

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Alrhmoun, S., O. Yu Perik-Zavodskaia, M. O. Volynets, Yu A. Lopatnikova, Yu A. Shevchenko, M. S. Fisher, R. Yu Perik‑Zavodskii, V. V. Kurilin, A. N. Silkov, and S. V. Sennikov. "OBTAINING FULL SEQUENCES OF CD8+ T-CELL RECEPTORS SPECIFIC TO THE TUMOR-ASSOCIATED ANTIGEN HER-2/NEU USING SINGLE CELL MRNA SEQUENCING AND ERGO-II NEURAL NETWORK." In OpenBio-2023. ИПЦ НГУ, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-9.

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The membrane protein HER-2/neu (Human Epidermal Growth Factor Receptor-2) is a tumor-associated antigen, the increased expression of which is observed in various types of tumors, and is associated, in particular, with the most aggressive types of breast cancer and poor clinical prognosis, which makes HER2/neu a universal target for the development of various immunotherapy approaches [1].

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Baker, J. B., M. P. McGrogan, C. Simonsen, R. L. Gronke, and B. W. Festoff. "STRUCTURE AND PROPERTIES OF PROTEASE NEXIN I." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644765.

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Human foreskin fibroblasts secrete several different serine protease inhibitors which differ in size and protease specificities. These proteins, called protease nexins (PNs) all form SDS-resistant complexes with their protease targets. Fibroblast surface receptors recognize the protease-PN complexes and mediate their delivery to lysosomes. PNI is a 45 kilodalton glycoprotein that rapidly inhibits several arg or lys-specific proteases including trypsin, thrombin, and urokinase (k assoc.∼ 4×l06,∼ 6×105 and ∼ 2×105, m−1s−1 respectively). Like antithrombin III, PNI binds heparin and inhibits thrombin at a vastly accelerated rate in the presence of this glycoaminoglycan. Immunofluorescence studies show that in addition to secreting PNI foreskin fibroblasts carry this inhibitor on their surfaces. PNI cDNA has been cloned and sequenced. A mixed oligonucleotide probe derived from PNI N-terminal sequence was used to probe a foreskin fibroblast cDNA library constructed with λGT10. Identification of PNI cDNAs has been verified by sequencing and by expressing active PNI protein in mammalian cells. The full amino acid sequence of PNI, deduced from cDNA sequencing, is 392 residues long and has 30% homology to antithrombin III. An arg-ser pair 32 residues from the C-terminus of the inhibitor is proposed as the reactive center P1-P1 residues. In the hinge region a lys residue is present in a position occupied by a ginor glu residue in other serpins. PNI mRNA exists in 2 slightly different forms:One (αPNI) yields a thr-arg-ser sequence wherethe other βPNI) yields a thr-thr-gly-ser sequence. The presence of the appropriate splice acceptor sites in the genome indicates that these forms are generated from a single gene by alternative splicing. Expressed aPNI and 0PNI proteins both bind thrombin and urokinase. In foreskin fibroblaststhe α form of PNI mRNA predominates over the β form by about 2:1. In foreskin fibroblast cultures secreted PNI inhibits the mitogenic response to thrombin and regulate secreted urokinase. Purified PNI added to human fibrosarcoma (HT1080) cells inhibitsthe tumor cell-mediated destruction of extracellular matrix and transiently, but dramatically, inhibits tumor cell growth. PNI or PNI-like inhibitors may function at multiple physiological sites. The β form of PNI is virtually identical to a glia-derived neurite promoting factor, the cDNA for which has been recently cloned and sequenced by Gloor et al (1). The neurite outgrowth activity of PNI may result from inhibition of a thrombin-like protease that is associated with neurons, since a number of thrombin inhibitors stimulate neurite extension. Recent immunofluoresence experiments, carried out with D. Hantai (Inserm; Paris) demonstrate that anti-PNI antibody intensely stains neuromuscular synapses. In addition, a PNI-like inhibitor is associated with platelets. At low (0.5 nM <) 125I-thrombin concentrations formation of 125I-thrombin-platelet PNI complexes accounts for most of the specific binding of 125I-thrombin to platelets (2). Although the platelet-associated form of PNI is electrophoretically and immunologically indistinguishable from fibroblast PNI, it does not bind urokinase, suggesting that it may be distinct.(1) Gloor, S., K. Odink, J. Guenther, H. Nick, and D. Monard. (1986) Cell 47:687-693.(2) Gronke, R.S., B.L. Bergman, and J.B. Baker. (1987) J. Biol. Chem. (in press)

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Reports on the topic "Single cell mRNA sequencing"

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Oskolkov, Nikolay. Single Cell Data Analysis. Instats Inc., 2024. http://dx.doi.org/10.61700/23nmyqoprtfw01698.

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This one-day workshop provides a comprehensive overview of single-cell data analysis, focusing on the advanced computational tools and methodologies required to interpret cellular heterogeneity using single-cell RNA sequencing technologies. Participants will gain practical skills in preprocessing, clustering, and integrating datasets, leveraging powerful R and Python libraries, with an emphasis on biological inference and real-world applications in fields such as cancer research and immunology.

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Harouaka, Ramdane. Platform for Single-Cell Dual RNA Sequencing of Host-Pathogen Interactions. Office of Scientific and Technical Information (OSTI), October 2021. http://dx.doi.org/10.2172/1832283.

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Fung, N. DNA sequencing with capillary electrophoresis and single cell analysis with mass spectrometry. Office of Scientific and Technical Information (OSTI), March 1998. http://dx.doi.org/10.2172/348902.

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Hovav, Ran, Peggy Ozias-Akins, and Scott A. Jackson. The genetics of pod-filling in peanut under water-limiting conditions. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597923.bard.

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Pod-filling, an important yield-determining stage is strongly influenced by water stress. This is particularly true for peanut (Arachishypogaea), wherein pods are developed underground and are directly affected by the water condition. Pod-filling in peanut has a significant genetic component as well, since genotypes are considerably varied in their pod-fill (PF) and seed-fill (SF) potential. The goals of this research were to: Examine the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Detect global changes in mRNA and metabolites levels that accompany PF and SF. Explore the response of the duplicate peanut pod transcriptome to drought stress. Study how entire duplicated PF regulatory processes are networked within a polyploid organism. Discover locus-specific SNP markers and map pod quality traits under different environments. The research included genotypes and segregating populations from Israel and US that are varied in PF, SF and their tolerance to water deficit. Initially, an extensive field trial was conducted to investigate the effects of genotype, irrigation, and genotype X irrigation on PF and SF. Significant irrigation and genotypic effect was observed for the two main PF related traits, "seed ratio" and "dead-end ratio", demonstrating that reduction in irrigation directly influences the developing pods as a result of low water potential. Although the Irrigation × Genotype interaction was not statistically significant, one genotype (line 53) was found to be more sensitive to low irrigation treatments. Two RNAseq studies were simultaneously conducted in IL and the USA to characterize expression changes that accompany shell ("source") and seed ("sink") biogenesis in peanut. Both studies showed that SF and PF processes are very dynamic and undergo very rapid change in the accumulation of RNA, nutrients, and oil. Some genotypes differ in transcript accumulation rates, which can explain their difference in SF and PF potential; like cvHanoch that was found to be more enriched than line 53 in processes involving the generation of metabolites and energy at the beginning of seed development. Interestingly, an opposite situation was found in pericarp development, wherein rapid cell wall maturation processes were up-regulated in line 53. Although no significant effect was found for the irrigation level on seed transcriptome in general, and particularly on subgenomic assignment (that was found almost comparable to a 1:1 for A- and B- subgenomes), more specific homoeologous expression changes associated with particular biosynthesis pathways were found. For example, some significant A- and B- biases were observed in particular parts of the oil related gene expression network and several candidate genes with potential influence on oil content and SF were further examined. Substation achievement of the current program was the development and application of new SNP detection and mapping methods for peanut. Two major efforts on this direction were performed. In IL, a GBS approach was developed to map pod quality traits on Hanoch X 53 F2/F3 generations. Although the GBS approach was found to be less effective for our genetic system, it still succeeded to find significant mapping locations for several traits like testa color (linkage A10), number of seeds/pods (A5) and pod wart resistance (B7). In the USA, a SNP array was developed and applied for peanut, which is based on whole genome re-sequencing of 20 genotypes. This chip was used to map pod quality related traits in a Tifrunner x NC3033 RIL population. It was phenotyped for three years, including a new x-ray method to phenotype seed-fill and seed density. The total map size was 1229.7 cM with 1320 markers assigned. Based on this linkage map, 21 QTLs were identified for the traits 16/64 weight, kernel percentage, seed and pod weight, double pod and pod area. Collectively, this research serves as the first fundamental effort in peanut for understanding the PF and SF components, as a whole, and as influenced by the irrigation level. Results of the proposed study will also generate information and materials that will benefit peanut breeding by facilitating selection for reduced linkage drag during introgression of disease resistance traits into elite cultivars. BARD Report - Project4540 Page 2 of 10

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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.

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The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.

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Bacharach, Eran, and Sagar Goyal. Generation of Avian Pneumovirus Modified Clones for the Development of Attenuated Vaccines. United States Department of Agriculture, November 2008. http://dx.doi.org/10.32747/2008.7696541.bard.

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Abstract (one page maximum, single spaced), include: List the original objectives, as defined in the approved proposal, and any revisions made at the beginning or during the course of project: The main goal described in our original proposal has been the development of a molecular infectious clone of the avian metapneumovirus subtype B (aMPV-B) and the modification of this clone to create mutated viruses for the development of attenuated vaccines. The Achievements and Appendix/Part I sections of this report describes the accomplishments in creating such a molecular clone. These sections also contain the results of a longitudinal study that we made in Israel, demonstrating the infiltration of field strains of aMPV into vaccinated flocks and emphasizing the need for the development of better vaccines. We also describe our unexpected findings regarding the ability of aMPV to establish persistent infection in cell cultures. Although this direction of research was not described in the original proposal we feel that it is highly important for the understanding of aMPV pathogenesis. For example, this direction has provided us with evidence showing that aMPV replication can augment influenza replication. Moreover, we observed that viruses that were produced from chronically-infected cells show reduced ciliostasis. Accordingly, we carried vaccination trials using such viruses. In the original grant proposal we also offered that the American lab will clone and express immunomodulators in the context of an aMPV -based replicon that the Israeli lab has generated. However, as we reported in our annual reports, further analysis of this replicon by the Israeli lab has revealed that the level of expression achieved by this vehicle is relatively poor; thus, the American lab has focused on sequencing the genomes of different aMPV-C isolates that differ in their virulence (including vaccine strains). Achievements and Appendix/Part II sections of this report include the summary of this effort. Background to the topic: The aMPVs belong to the paramyxoviridae family and cause mild to severe respiratory tract diseases mainly in turkeys and also in chickens. Four aMPV subgroups, A, B, C and D, have been characterized; in Israel aMPV-A and B are the common subtypes while in the USA type C is the prevalent one. Although vaccine strains do exist for aMPVs, they do not always provide full protection against virulent strains and the vaccines themselves may induce disease to some extent. Improved vaccines against aMPV are needed, to achieve better protection of the poultry industry against this pathogen. Major conclusions, solutions, achievements: We isolated aMPV-B from a diseased flock and accomplished the sequencing and cloning of its full-genome. In addition, we cloned the four genes encoding the viral replicase. These should serve as the platform for generation of modified aMPV-Bs from molecular clones. We also identified aMPVs that are attenuated in respect to their ciliostatic activity and accordingly showed the potential of such viruses as vaccine strains. For aMPV-C, the different mutations scattered along the genome of different isolates with varied virulence have been determined. Implications, both scientific and agricultural: The newly identified pattern of mutations in attenuated strains will allow better understanding of the pathogenicity of aMPV and the generation of aMPV molecular clones, together with isolation of strains with attenuated ciliostatic activity should generate improved vaccine strains Abstract (one page maximum, single spaced), include: List the original objectives, as defined in the approved proposal, and any revisions made at the beginning or during the course of project: The main goal described in our original proposal has been the development of a molecular infectious clone of the avian metapneumovirus subtype B (aMPV-B) and the modification of this clone to create mutated viruses for the development of attenuated vaccines. The Achievements and Appendix/Part I sections of this report describes the accomplishments in creating such a molecular clone. These sections also contain the results of a longitudinal study that we made in Israel, demonstrating the infiltration of field strains of aMPV into vaccinated flocks and emphasizing the need for the development of better vaccines. We also describe our unexpected findings regarding the ability of aMPV to establish persistent infection in cell cultures. Although this direction of research was not described in the original proposal we feel that it is highly important for the understanding of aMPV pathogenesis. For example, this direction has provided us with evidence showing that aMPV replication can augment influenza replication. Moreover, we observed that viruses that were produced from chronically-infected cells show reduced ciliostasis. Accordingly, we carried vaccination trials using such viruses. In the original grant proposal we also offered that the American lab will clone and express immunomodulators in the context of an aMPV -based replicon that the Israeli lab has generated. However, as we reported in our annual reports, further analysis of this replicon by the Israeli lab has revealed that the level of expression achieved by this vehicle is relatively poor; thus, the American lab has focused on sequencing the genomes of different aMPV-C isolates that differ in their virulence (including vaccine strains). Achievements and Appendix/Part II sections of this report include the summary of this effort. Background to the topic: The aMPVs belong to the paramyxoviridae family and cause mild to severe respiratory tract diseases mainly in turkeys and also in chickens. Four aMPV subgroups, A, B, C and D, have been characterized; in Israel aMPV-A and B are the common subtypes while in the USA type C is the prevalent one. Although vaccine strains do exist for aMPVs, they do not always provide full protection against virulent strains and the vaccines themselves may induce disease to some extent. Improved vaccines against aMPV are needed, to achieve better protection of the poultry industry against this pathogen. Major conclusions, solutions, achievements: We isolated aMPV-B from a diseased flock and accomplished the sequencing and cloning of its full-genome. In addition, we cloned the four genes encoding the viral replicase. These should serve as the platform for generation of modified aMPV-Bs from molecular clones. We also identified aMPVs that are attenuated in respect to their ciliostatic activity and accordingly showed the potential of such viruses as vaccine strains. For aMPV-C, the different mutations scattered along the genome of different isolates with varied virulence have been determined. Implications, both scientific and agricultural: The newly identified pattern of mutations in attenuated strains will allow better understanding of the pathogenicity of aMPV and the generation of aMPV molecular clones, together with isolation of strains with attenuated ciliostatic activity should generate improved vaccine strains.

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Hansen, Peter J., and Zvi Roth. Use of Oocyte and Embryo Survival Factors to Enhance Fertility of Heat-stressed Dairy Cattle. United States Department of Agriculture, August 2011. http://dx.doi.org/10.32747/2011.7697105.bard.

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The overall goal was to identify survival factors that can improve pregnancy success following insemination or embryo transfer in lactating dairy cows exposed to heat stress. First, we demonstrated that oocytes are actually damaged by elevated temperature in the summer. Then we tested two thermoprotective molecules for their effect on oocyte damage caused by heat shock. One molecule, ceramide was not thermoprptective. Another, insulin-like growth factor-1 (IGF) reduced the effects of heat shock on oocyte apoptosis and oocyte cleavage when added during maturation. We also used lactating cows exposed to heat stress to determine whether bovine somatotropin (bST), which increases IGF1 levels in vivo, would improve fertility in summer. Cows treated with bST received a single injection at 3 days before insemination. Controls received no additional treatment. Treatment with bST did not significantly increase the proportion of inseminated cows diagnosed pregnant although it was numerically greater for the bST group (24.2% vs 17.8%, 124–132 cows per group). There was a tendency (p =0.10) for a smaller percent of control cows to have high plasma progesterone concentrations (≥ 1 ng/ml) at Day 7 after insemination than for bST-treated cows (72.6 vs 81.1%). When only cows that were successfully synchronized were considered, the magnitude of the absolute difference in the percentage of inseminated cows that were diagnosed pregnant between bST and control cows was reduced (24.8 vs 22.4% pregnant for bST and control). Results failed to indicate a beneficial effect of bST treatment on fertility of lactating dairy cows. In another experiment, we found a tendency for addition of IGF1 to embryo culture medium to improve embryonic survival after embryo transfer when the experiment was done during heat stress but not when the experiment was done in the absence of heat stress. Another molecule tested, granulocyte-macrophage colony-stimulating factor (GM-CSF; also called colony-stimulating factor-2), improved embryonic survival in the absence of heat stress. We also examined whether heat shock affects the sperm cell. There was no effect of heat shock on sperm apoptosis (programmed cell death) or on sperm fertilizing ability. Therefore, effects of heat shock on sperm function after ejaculation if minimal. However, there were seasonal changes in sperm characteristics that indicates that some of the decrease in dairy cow fertility during the summer in Israel is due to using semen of inferior quality. Semen was collected from five representative bulls throughout the summer (August and September) and winter (December and January). There were seasonal differences in ion concentration in seminal plasma and in the mRNA for various ion channels known to be involved in acrosome reactions. Furthermore, the proportion of sperm cells with damaged acrosomes was higher in post-thaw semen collected in the summer than in its counterpart collected in winter (54.2 ± 3.5% vs. 51.4 ± 1.9%, respectively; P < 0.08Further examination is required to determine whether such alterations are involved in the low summer fertility of dairy cows.

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Palmer, Guy, Varda Shkap, Wendy Brown, and Thea Molad. Control of bovine anaplasmosis: cytokine enhancement of vaccine efficacy. United States Department of Agriculture, March 2007. http://dx.doi.org/10.32747/2007.7695879.bard.

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Anaplasmosis an arthropod-born disease of cattle caused by the rickettsia Anaplasma marginale and is an impediment to efficient production of healthy livestock in both Israel and the United States. Currently the only effective vaccines are derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Consequently development of a safe, effective vaccine is a high priority. In this collaborative project we focused on two approaches to vaccine development. The first focused o n improving antigen delivery to livestock and specifically examined how DNA vaccines could be improved to enhance priming and expansion of the immune response. This research resulted in development and testing of two novel vaccine delivery systems--one that targeted antigen spread among dendritic cells (the key cell in priming immune responses and a follow-on construct that also specifically targeted antigen to the endosomal-lysosomal compartment the processing organelle within the dendritic cell that directs vaccine antigen to the MHC class ll-CD4* T cell priming pathway). The optimized construct targeting vaccine antigen to the dendritic cell MHC class II pathway was tested for ability to prime A. marginale specific immune responses in outbred cattle. The results demonstrated both statistically significant effects of priming with a single immunization, continued expansion of the primary immune response including development of high affinity lgG antibodies and rapid recall of the memory response following antigen challenge. This portion of the study represented a significant advance in vaccine delivery for livestock. Importantly the impact of these studies is not limited to A. marginale a s the targeting motifs are optimized for cattle and can be adapted to other cattle vaccinations by inserting a relevant pathogen-specific antigen. The second approach (which represented an addition to the project for which approval was requested as part of the first annual report) was a comparative approach between A . marginale and the Israel A . centrale vaccines train. This addition was requested as studies on Major Surface Protein( MSP)- 2 have shown that this antigen is highly antigenically variable and presented solely as a "static vaccine" antigen does not give cross-strain immunity. In contrast A. . centrale is an effective vaccine which Kimron Veterinary institute has used in the field in Israel for over 50 years. Taking advantage of this expertise, a broad comparison of wild type A. marginale and vaccine strain was initiated. These studies revealed three primary findings: i) use of the vaccine is associated with superinfection, but absence of clinical disease upon superinfection with A. marginale; ii) the A. centrale vaccine strain is not only less virulent but transmission in competent in Dermacentor spp. ticks; and iii) some but not all MSPs are conserved in basic orthologous structure but there are significant polymorphisms among the strains. These studies clearly indicated that there are statistically significant differences in biology (virulence and transmission) and provide a clear path for mapping of biology with the genomes. Based on these findings, we initiated complete genome sequencing of the Israel vaccine strain (although not currently funded by BARD) and plant to proceed with a comparative genomics approach using already sequenced wild-type A. marginale. These findings and ongoing collaborative research tie together filed vaccine experience with new genomic data, providing a new approach to vaccine development against a complex pathogen.

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Fahima, Tzion, and Jorge Dubcovsky. Map-based cloning of the novel stripe rust resistance gene YrG303 and its use to engineer 1B chromosome with multiple beneficial traits. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598147.bard.

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Research problem: Bread wheat (Triticumaestivum) provides approximately 20% of the calories and proteins consumed by humankind. As the world population continues to increase, it is necessary to improve wheat yields, increase grain quality, and minimize the losses produced by biotic and abiotic stresses. Stripe rust, caused by Pucciniastriiformisf. sp. tritici(Pst), is one of the most destructive diseases of wheat. The new pathogen races are more virulent and aggressive than previous ones and have produced large economic losses. A rich source for stripe-rust resistance genes (Yr) was found in wild emmer wheat populations from Israel. Original Project goals: Our long term goal is to identify, map, clone, characterize and deploy in breeding, novel wild emmer Yr genes, and combine them with multiple beneficial traits. The current study was aiming to map and clone YrG303 and Yr15, located on chromosome 1BS and combine them with drought resistance and grain quality genes. Positional cloning of YrG303/Yr15: Fine mapping of these genes revealed that YrG303 is actually allelic to Yr15. Fine genetic mapping using large segregating populations resulted in reduction of the genetic interval spanning Yr15 to less than 0.1 cM. Physical mapping of the YrG303/Yr15 locus was based on the complete chromosome 1BS physical map of wheat constructed by our group. Screening of 1BS BAC library with Yr15 markers revealed a long BAC scaffold covering the target region. The screening of T. dicoccoidesaccession-specific BAC library with Yr15 markers resulted in direct landing on the target site. Sequencing of T. dicoccoidesBAC clones that cover the YrG303/Yr15 locus revealed a single candidate gene (CG) with conserved domains that may indicate a role in disease resistance response. Validation of the CG was carried out using EMS mutagenesis (loss-of- function approach). Sequencing of the CG in susceptible yr15/yrG303 plants revealed three independent mutants that harbour non-functional yr15/yrG303 alleles within the CG conserved domains, and therefore validated its function as a Pstresistance gene. Evaluation of marker-assisted-selection (MAS) for Yr15. Introgressions of Yr15 into cultivated wheat are widely used now. Recently, we have shown that DNA markers linked to Yr15 can be used as efficient tools for introgression of Yr15 into cultivated wheat via MAS. The developed markers were consistent and polymorphic in all 34 tested introgressions and are the most recommended markers for the introgression of Yr15. These markers will facilitate simultaneous selection for multiple Yr genes and help to avoid escapees during the selection process. Engineering of improved chromosome 1BS that harbors multiple beneficial traits. We have implemented the knowledge and genetic resources accumulated in this project for the engineering of 1B "super-chromosome" that harbors multiple beneficial traits. We completed the generation of a chromosome including the rye 1RS distal segment associated with improved drought tolerance with the Yr gene, Yr15, and the strong gluten allele 7Bx-over-expressor (7Bxᴼᴱ). We have completed the introgression of this improved chromosome into our recently released variety Patwin-515HP and our rain fed variety Kern, as well as to our top breeding lines UC1767 and UC1745. Elucidating the mechanism of resistance exhibited by Yr36 (WKS1). The WHEAT KINASE START1 (WKS1) resistance gene (Yr36) confers partial resistance to Pst. We have shown that wheat plants transformed with WKS1 transcript are resistant to Pst. WKS1 is targeted to the chloroplast where it phosphorylates the thylakoid-associatedascorbateperoxidase (tAPX) and reduces its ability to detoxify peroxides. Based on these results, we propose that the phosphorylation of tAPX by WKS1 reduces the ability of the cells to detoxify ROS and contributes to cell death. Distribution and diversity of WKS in wild emmer populations. We have shown that WKS1 is present only in the southern distribution range of wild emmer in the Fertile Crescent. Sequence analysis revealed a high level of WKS1 conservation among wild emmer populations, in contrast to the high level of diversity observed in NB-LRR genes. This phenomenon shed some light on the evolution of genes that confer partial resistance to Pst. Three new WKS1 haplotypes displayed a resistance response, suggesting that they can be useful to improve wheat resistance to Pst. In summary, we have improved our understanding of cereals’ resistance mechanisms to rusts and we have used that knowledge to develop improved wheat varieties.

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