Progress 06/01/20 to 05/31/24
Outputs
Target Audience:The ongoing intense interest in and need for improvement of the current linear versions of the chicken genome reference, as well as the development of an accompanying catalog of annotated immune cell types useful for poultry disease studies, led us to develop each resource to serve a broad scope of research interests. Our premise is that increasing the number of high-quality genomic references that better represent commercial chicken lines will improve future study outcomes when examining how pathogen changes affect all commercial chicken populations. The culmination of our resource project addresses these research community needs by providing significantly improved versions of the chicken genome, specifically a representation of broiler and layer birds rather than the historical red jungle fowl, resulting better immune gene families annotation, a first single-cell atlas focused on immune cell types derived from the chicken's major immune organ systems of spleen, bursa, and thymus, and a multifaceted genome browser to simplify access and queries of all cell type gene expression data. Overall, we met our objectives by communicating descriptions of these new resources and subsequent research advances to selected audiences such as poultry business representatives, academic researchers, and USDA scientists, as well as publishing our findings. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Our lab has been incredibly productive and impactful in advancing genomics research, particularly in the avian field. The successful implementation and widespread adoption of our updated chicken reference resources, especially the broiler version, underscores the value of this work in the community. By providing both the tools and the training, we are equipping researchers with the skills needed to leverage these resources fully. With easy access to our new chicken reference resources the community is utilizing the broiler version as a linear reference for all purposes. One example would be in the use of these references and other assembled chicken genomes was our build of a pangenome reference that shows the advantages over linear references. We produced a report that details the computational methods used to generate pangenome graphs of chicken genomes, which can be used as a guideline for creating one's own chicken pangenome graph as well as sharing our experience for other agricultural species applications. Other groups, for instance the horse pangenome consortium, have contacted us with detailed queries about how to establish the software parameters for pangenome builds. All these genome reference uses showcase training. In addition, we have used single nuclei RNAseq data to train graduate students, postdocs, and faculty on the all processes related to its interpretation toward a goal of well curated cell types. One MS student trained and published her findings that led to admittance into an Animal Sciences PhD program. Internally, we exposed computational labs to the process of setting up portals for scientists to view gene expression data by cell type in previously established browsers. Our recent on-line training on the process of annotating immune cell types with members of the avian immunology community has been informative for all. Through our collaborative efforts, many members of the avian research community have been exposed for the first time to evaluating snRNAseq data, including its strengths and drawbacks. How have the results been disseminated to communities of interest?As noted earlier we have submitted the layer and broiler assemblies to NCBI for public use and importantly files that provide the predicted protein-coding genes associated with each reference are now accessible. In the products section of this report, we describe how these resources are downloadable and can be queried for sequences of interest via the NCBI genome viewer. For all single nuclei transcriptome data generated, we have submitted these sequences if they passed quality checks to the NCBI sequence read archive in accordance with our data sharing plan. New sequences generated at this end of this funding period for live immune cells, not nuclei, will be disseminated the same as for prior seqeuncing data. By presenting multiple research progress updates to the avian genomics community, industry scientists, and others at several meetings over the course of this funding period including AGBT AG, Plant and Animal Genomes, and USDA sponsored workshops their understanding of the data utility was heightened. As noted in the products section. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Our primary objective was to offer novel sources of information for commercially relevant versions of the broiler and layer genome. During our last year, we collaborated with colleagues from the Vertebrate Genome Project to enhance the accuracy of both references. Our goal is to replace the current chicken genome reference, GRCg7b. To utilize the most up-to-date methods for de novo assembly, we collected long-read data from two different platforms, Oxford Nanopore Technology and Pacific Biosciences. This allowed us to create a new version of the chicken genome reference that spans from telomere to telomere (T2T). We expect the T2T broiler and layer genome assemblies, currently in progress, to be the most comprehensive and accurate references to date. Future computational investigations increasingly view adopting T2T versions of all chromosomes as the benchmark for achieving high quality. Furthermore, the use of these T2T versions as a reference template for the construction of a pangenome is highly valuable. To offer the community, the most comprehensive genomic resource for chicken, we have utilized a combination of long-read assemblies to create the first full pangenome graph of chicken. This initial chicken pangenome graph includes 30 DNA samples of different types of broilers, layers, trait selected research lines, and selected breeds. Importantly, we have consistently utilized the same chicken broiler/layer DNA samples used in generating earlier long-read data sets, and adhering to similar graph build criteria as the pig and cow pangenome groups. Our pangenome reference showcases the future of genomic reference use. All throughout the duration of this proposal, the improved broiler and layer linear genome references were available for download from the NCBI and Ensembl genome browsers. To achieve our second primary goal, we collected and cryopreserved all targeted avian tissue samples, honed our nuclei isolation techniques, and generated data through a technique known as single nuclei RNA sequencing (snRNAseq). Although we faced considerable technical challenges in isolating nuclei of superior quality from different chicken tissues, we managed to overcome these obstacles thanks to recent advancements in single-cell technology techniques. In addition, we conducted a treated vs. control experiment to elucidate the computational approaches that underly the interpretation of snRNAseq results for a broad audience of avian biologists. We obtained samples of the spleen and bursa, after subjecting chickens in vivo to a mimic of bacterial infection known as lipopolysaccharide. From the standpoint of the host's overall immune response, we have produced a detailed account in our published findings. As part of our efforts, we have focused on developing and implementing bioinformatic pipelines to process pangenome and snRNA sequencing data in chickens. We formed a team of avian immunology experts to curate a cell type and develop a procedure to address this issue. A goal to find gene markers for the main immune cell types in the bursa, thymus, and spleen was met that will be valuable for future studies in avian immunology. To accomplish our third resource objective, we have created a website called the Chicken Cell Atlas for open access to cell type specific molecular profiles. This website was constructed using the knowledge obtained from groups that have developed cell atlas genome browsers for various species, such as the Cell Browser provided by the University of California, Santa Cruz. This report's products section contains more information about this website. The navigation bar in our chicken cell atlas browser contains hyperlinks to both the genome browser and the cell browser. This resource will enable more comprehensive curation of chicken immune cell types in the future.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Rice ES, Alberdi A, Alfieri J, Athrey G, Balacco JR, Bardou P, Blackmon H, Charles M, Cheng HH, Fedrigo O, Fiddaman SR, Formenti G, Frantz LAF, Gilbert MTP, Hearn CJ, Jarvis ED, Klopp C, Marcos S, Mason AS, Velez-Irizarry D, Xu L, Warren WC. 2023. A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants. BMC Biol. Nov 22;21(1):267.
- Type:
Journal Articles
Status:
Published
Year Published:
2024
Citation:
Elaina R. Sculley, Edward S. Ricemeyer, Rachel C. Carroll, John Driver, Jacqueline Smith, Jim Kaufman, Cari Hearn, Adam Balic, Paula Chen, Susan Lamont, Skyler Kramer, Yvonne Drechsler, Hans Cheng, Wesley C. Warren. 2024. A single-nucleus census of immune and non-immune cell types for the major immune organ systems of chicken. bioRxiv 08.05.606414; doi: https://doi.org/10.1101/2024.08.05.606414.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Warren WC, Rice SR, Meyer A, Hearn CJ, Stepp A, Hunt HD, Monson MS, Lamont SJ, Cheng HH. 2023. The immune cell landscape and response of Mareks disease resistant and susceptible chickens infected with Mareks disease virus. Sci Rep. 2023 Apr 1;13(1):5355. doi: 10.1038/s41598-023-32308-x. PMID: 37005445.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Smith J, et al. 2023. Fourth report on chicken genes and chromosomes 2023. Cytogenet Genome Res. Jan 30 online ahead of print. PMID: 36716736.
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Progress 06/01/22 to 05/31/23
Outputs
Target Audience:A continued intense interest in and need for improvement of the current version of the chicken genome reference as well as the development of an accompanying catalog of annotated immune cell types useful for poultry disease studies led us to target a primary audience of industry but additionally academia. Our expectation is the expanded use of multiple high quality genome references that better represents commercial chicken lines will improve future studies outcomes when assessing how fluctuations in the pathogen environment effect all commercial populations of chicken. Our resource project addresses these research community needs by: providing near complete and accurate versions of the chicken genome for broiler and layer birds, in particular better immune gene families annotation, a first for representative layer and broiler genetic lines, a single-cell atlas enriched for immune cell types that catalog cell markers for the chicken's major immune organ systems and in later project stages presentation of a multifaceted genome browser to simplify access and queries of all data generated. In this third year, we have communicated our resource and research progress to poultry industry representatives, the academic researchers, USDA scientists, and published our findings. Changes/Problems:This research experienced a one-year delay due to the COVID-19 pandemic, but with the adjusted timelines, we remain closely aligned with our original goals. The only significant issue we have faced is the need to revise our standard nuclei isolation techniques for thymus tissue, as mentioned earlier. Our current progress is outlined in the "What You Plan to Do" section above, and we are confident that we will overcome any remaining technical challenges. Notably, using new debris removal buffers our nuclei counts and staining results are promising, suggesting that we will be ready to proceed with final snRNAseq data generation within the next few weeks. What opportunities for training and professional development has the project provided?With ongoing access to our newly developed chicken references, pangenome graph, and cell-type-specific molecular profiles, we anticipate that the broader scientific community using chickens as a model organism will be able to test a wide range of hypotheses regarding phenotype differences. These resources will also help researchers uncover the molecular mechanisms behind traits critical to bird health. By leveraging new insights into gene expression patterns by cell type, these data will serve as a computational resource for numerous research applications. For example, by using the gene expression profiles that define certain cell types we can train personnel how to deconvolute aggregate RNAseq data to the underlaying cell types per sample. A valuable approach to discover gene regulatory changes that help explain phenotypic differences in commercial chicken populations. These genetic tools will also provide valuable training opportunities for graduate students, postdocs, and faculty, helping them to master and refine new computational techniques. One of our graduate students recently completed her MS by utilizing our snRNAseq datasets. Our recent efforts to train members of the avian immunology community in immune cell type annotation have been highly informative. For many in the community, this collaboration marks their first exposure to interpreting snRNAseq data, giving them a deeper understanding of its strengths and limitations. How have the results been disseminated to communities of interest?As noted earlier we have submitted the layer and broiler assemblies to NCBI for public use and importantly files that provide the predicted protein-coding genes associated with each reference are now accessible. In the products section of this report, we describe how these resources are downloadable and can be queried for sequences of interest via the NCBI genome viewer. For all single nuclei transcriptome data generated, we have submitted these sequences if they passed quality checks to the NCBI sequence read archive in accordance with our data sharing plan. As new sequences are generated, we plan to follow these same steps and make the data available as soon as possible following various phases of quality filtering and interpretation. What do you plan to do during the next reporting period to accomplish the goals?We are continually refining our nuclei preparation protocols for tissue types that have yet to be profiled, aiming to provide the community with the best possible guidance for generating high-quality data. The thymus, for example, is a tissue where further optimization of our current protocols could significantly enhance the results we've obtained so far. Our primary goal for the coming year is to publish the first comprehensive census of immune cell types across the major immune organs of the chicken. Once we complete our investigation into cell type variability across different organ systems, we will explore innovative ways to share this data with the community. One approach will involve performing gene trajectory analysis that is independent of cell type, adding depth to our current interpretations of cell populations. We anticipate uncovering many intriguing insights into both innate and adaptive immune cells, particularly in how they respond to pathogen challenges.
Impacts
What was accomplished under these goals?
We have three critical goals to enable future studies of periodic pathogen challenges experienced in the poultry industry: establish high-quality genome references that are more representative of commercial birds, broilers and layers to test pangenome graph outcomes, generate a single-cell atlas enriched for immune cell types with accompanying catalog of cell-type specific markers and the build of a multifaceted genome browser to simplify access and queries of all cell atlas data generated. In this third year, we have completed most of our objectives in this year's funding period. In this year, a collaboration with members of the Vertebrate Genome Project led to the further improvement in the quality of the broiler and layer individual assemblies by extensive curation of detected assembly errors in each and their manual correction. Both are available on all the major genome browsers. Toward our second objective, we have completed the collection of all bird tissue samples targeted, and then developed successfully executed nuclei isolation protocols for all samples. Additionally, we have completed our evaluation of host response to Marek's disease virus at single cell resolution in the spleen and published our findings. Currently, we are in the final phases of analyzing the single nuclei RNA sequencing (snRNAseq) data generated from spleen, bursa, and thymus tissues to develop a immune cell type focused atlas resource for the community use. We also exposed some birds in vivo to a bacterial mimic of infection (LPS) to demonstrate the powerful discovery potential of snRNAseq data. A manuscript describing these LPS treatment results in the bursa is drafted and under review by our team. A significant part of our progress has involved the testing and implementation of bioinformatic pipelines for processing snRNAseq data in chicken, including the establishment of a cell type curation team comprised of experts in avian immunology. Through this process, our team has identified gene markers for major immune cell types in the known organ systems that support host responses to pathogen challenges. Our findings will be published to allow the community to compare and debate which gene markers are most relevant for the study of host response to pathogens. For our third objective, in collaboration with teams that have developed specialized human genome browsers, such as the Chan Zuckerberg CELL by GENE Discover database, we have set up portals to allow future, more detailed curation of chicken immune cell types. Furthermore, our collaboration with USDA scientists has helped us improve the annotation of immune cell types from spleen, bursa, and lung snRNAseq data
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Warren WC, Rice SR, Meyer A, Hearn CJ, Stepp A, Hunt HD, Monson MS, Lamont SJ, Cheng HH. 2023. The immune cell landscape and response of Mareks disease resistant and susceptible chickens infected with Mareks disease virus. Sci Rep. 2023 Apr 1;13(1):5355
- Type:
Journal Articles
Status:
Accepted
Year Published:
2023
Citation:
Smith J, et al. 2023. Fourth report on chicken genes and chromosomes 2023. Cytogenet Genome Res
|
Progress 06/01/21 to 05/31/22
Outputs
Target Audience:A continued intense interest in and need for improvement of the current version of the chicken genome reference as well as the development of an accompanying catalog of annotated immune cell types useful for poultry disease studies led us to target a primary audience of industry but additionally academia. Our expectation is the expanded use of multiple high quality genome references that better represents commercial chicken lines will improve future studies outcomes when assessing how fluctuations in the pathogen environment effect all commercial populations of chicken. Our resource project addresses these research community needs by: providing near complete and accurate versions of the chicken genome for broiler and layer birds, in particular better immune gene families annotation, a first for representative layer and broiler genetic lines, a single-cell atlas enriched for immune cell types that catalog cell markers for the chicken's major immune organ systems and in later project stages presentation of a multifaceted genome browser to simplify access and queries of all data generated. In this third year, we have communicated our resource and research progress to poultry industry representatives, the academic researchers, USDA scientists, and published our findings. Changes/Problems:Since this research was delayed by a full year due to the covid pandemic at the start we are still closely aligned to our originally set timelines assuming a delay adjustment. Our only major problem to report has been the need to revise our standard nuclei isolation techniques for the thymus tissue as noted earlier in this report. We describe our status in the what you plan to do section above. We are confident we will overcome this technical hurdle encountered with experiments underway. In fact, we recently obtained nuclei counts and staining data that suggests we can plan our thymus snRNAseq data generation in the next few weeks. What opportunities for training and professional development has the project provided?With easy access to our new chicken reference resources, we expect the broader community using chicken as a model organism or to understand molecular mechanisms of action for numerous traits of importance to bird health to use them as a computational conduit in many research applications. One example would be to map a set of resequenced chicken populations to these references for improved sequence variant ascertainment because of fewer erroneously mapped reads and better reference sequence representation with fewer gaps. These references will serve to train numerous graduate students, postdocs, and faculty who wish to learn and refine new computational techniques as well. When starting structural variant experiments by cross-referencing the long reads associated with each reference new breakthroughs can be made. Our recent training on the process of annotating immune cell types with members of the avian immunology community has been informative for all. Many in the avian research community through our collaborative efforts have for the first time been exposed to interpreting snRNAseq data and all its strengths and weaknesses. How have the results been disseminated to communities of interest?As noted earlier we have submitted the layer and broiler assemblies to NCBI for public use and importantly files that provide the predicted protein-coding genes associated with each reference are now accessible. In the products section of this report, we describe how these resources are downloadable and can be queried for sequences of interest via the NCBI genome viewer. For all single nuclei transcriptome data generated, we have submitted these sequences if they passed quality checksr to the NCBI sequence read archive in accordance with our data sharing plan. As new sequences are generated, we plan to follow these same steps and make the data available as soon as possible following various phases of quality filtering and interpretation. What do you plan to do during the next reporting period to accomplish the goals?We continue to optimize nuclei preparation procedures on the tissue type not profiled to date, the thymus. The thymus requires the additional testing of various iterations of our existing protocols as well as recently published procedures since thus far the snRNAseq quality has not been acceptable. Our immediate goal from this work is to choose the best nuclei procedure specific to thymus and complete the sequencing of nuclei from this tissue in the next few months. All snRNAseq data will be processed to collate all gene expression markers that define immune cell types across various tissue sources. A graduate student will be using the snRNAseq data in hand to explore many aspects of the resident cell types in the spleen and publish their findings. Shortly after completing the spleen analysis, we will analyze the bursa snRNAseq data set to reveal all the fascinating features of different B cell types that are present and respond to bacterial infection. For these and other collected immune organ sources we plan to publish our findings toward the creation of a comprehensive atlas of all chicken immune cell types.
Impacts
What was accomplished under these goals?
Our first objective was to generate new references for a representative broiler and layer genome. In this second year, in collaboration with members of the Vertebrate Genome Project we have accomplished this objective. A broiler and layer genome reference are now available for download at the NCBI and Ensembl genome browsers. These references are highly accurate and have now placed sequences to some microchromosomes that were missing in the current chicken reference, GRCg6a. Gene annotation is complete for both references with a substantial increase in the number of annotated non-coding genes discovered. Toward our second objective, we have completed the collection of all bird tissue samples targeted in this study, developed, and executed nuclei isolation protocols for all. In addition, we have completed and published a single cell RNAseq analysis of the chicken spleen when challenged with Marek's virus. The analysis of single nuclei RNA sequencing (snRNAseq) data generated for spleen and bursa exposed in vivo to a bacterial mimic of infection (LPS) is underway for spleen and bursa organ systems with intent to publish this year. A large part of our accomplishments has been the testing and implementation of bioinformatic pipelines for processing the snRNAseq data in chicken including the establishment of a cell type curation team comprised of experts in avian immunology. Using this process our team has discovered gene markers for the major immune cell types in the known organ systems that support host response to pathogen challenge. To make progress on our third objective, in collaboration with teams that have developed specialized human genome browsers, for example the Chan Zuckerberg CELL by GENE Discover database, we have set up portals to allow future curation of chicken immune cell types in greater detail. In addition, we have used our collaboration with UDSA scientists to learn how to better annotate immune cell types from spleen, bursa, and lung snRNAseq data.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Smith J., et al. Fourth Report on Chicken Genes and Chromosomes 2022. Cytogenet Genome Res. Published online: January 30th, 2023.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Warren W.C., et al. 2023. The immune cell landscape and response of Mareks disease resistant and susceptible chickens infected with Mareks disease virus. Sci Reports.
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