Source: WESTERN UNIVERSITY OF HEALTH SCIENCES submitted to NRP
GENOME-WIDE ANNOTATION OF CIS-REGULATORY ELEMENTS IN THE CHICKEN GENOME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1014871
Grant No.
2018-67015-27499
Cumulative Award Amt.
$2,500,000.00
Proposal No.
2017-05205
Multistate No.
(N/A)
Project Start Date
Mar 1, 2018
Project End Date
Feb 28, 2025
Grant Year
2019
Program Code
[A1202]- Annotation of Agricultural Animal Genomes
Recipient Organization
WESTERN UNIVERSITY OF HEALTH SCIENCES
309 EAST SECOND STREET
POMONA,CA 91766
Performing Department
(N/A)
Non Technical Summary
Infection and disease adversely impact performance, growth, morbidity and mortality in production animals and has major economic impacts on commercial poultry production. This is exacerbated by consumer worries about antibiotic use, which pressures producers to use alternative strategies to prevent infection. An illustrating example is the avian influenza outbreak of 2015, which has been described as the worst in US history with the loss of over 48 million birds (chicken and turkey). This event underscores the need to functionally annotating the chicken genome to understand complex genetic traits such as disease resistance, which in turn could serve as a powerful tool to ensure sustainable agricultural systems. Furthermore, in poultry production, additional factors affecting commercial and economic performance need to be considered in breeding. Factors including intestinal health, egg laying performance or growth efficiency in broilers certainly are of particular economic significance.To improve our understanding of the underlying mechanisms controlling these and other traits, identification of cis-regulatory elements on the genome is warranted. These regulatory elements control the expression of genes and consequently phenotype; however, the targeted tissues and cells for analysis need to be determined for each trait of interest, as these regulatory elements are tissue specific, and even differ from cell to cell within a given tissue. Additionally, environmental factors such as nutrition interact with epigenetic modifiers working at these regulatory elements, thus making it possible to promote the expression of desirable productive traits by management.In the past, selective breeding of poultry has resulted in improved feed conversion ratios in broilers, albeit at the cost of decreased immune competence and greater susceptibility to disease. In contrast, some lines of birds are known to be more resistant to a variety of pathogens.The emergence of antimicrobial resistance suggests that genetic resistance to disease would provide an advantage over the use of antimicrobials. While parameters of immune competence, such as antibody production, phagocytic activity and cytokine production of the host are known, the genetic and molecular basis for disease resistance is still not well understood. It has become apparent that pathogens interact with epigenetic regulators of the host, aiding immune evasion and replication of the pathogen.Identification of regulatory elements and their influence on target genes directly responsible for enhanced immunity in poultry is crucial in order to select for traits that improve disease resistance for breeding purposes and flock health.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30432991080100%
Knowledge Area
304 - Animal Genome;

Subject Of Investigation
3299 - Poultry, general/other;

Field Of Science
1080 - Genetics;
Goals / Objectives
Gene regulatory element annotation. The ENCODE project was launched more than a decade ago with the goal of better annotating the human genome for both coding and noncoding elements. This has greatly improved our understanding of gene regulation and dramatically advanced our understanding of disease-associated genetic variants. Applying this approach to annotate the chicken genome will catapult research in agricultural animals. Epigenetic modifications such as histone tail modifications and DNA methylation are not only key to the regulation of unique transcriptome patterns, these modifications are indispensable as genome annotators to uncover cell- and tissue-specific regulatory elements.Uncovering the location of regulatory elements and determining their interactions will provide the necessary framework to understand how regulatory networks govern gene expression and how genetic and environmental influences alter these networks to impact animal growth, health and disease susceptibility or resistance.The goal of this proposal is to leverage transcriptomic and epigenomic methods to annotate transcripts - coding and noncoding; and cis-regulatory elements in the chicken genome, including promoters, enhancers, silencers and insulators. Temporal, spatial, and tissue-specific gene expression is dependent on cis-regulatory elements. Providing such annotations to the agricultural community will have a profound impact on how chicken health and disease are studied. It will provide a reference for understanding gene regulation through the physical location of regulatory elements as well as reference epigenomes for 20 cell and tissue types that we have proposed. Furthermore, a more complete annotation of the chicken genome will enhance the investigation of genetic traits as most associated genetic variants are likely to reside outside of genes and within noncoding elements. Similar mapping endeavors in other animals such as human and mouse have revealed more noncoding regulatory elements than the number of genes they regulate. Collectively, these elements orchestrate complex, but well choreographed three-dimensional interactions in the genome to control expression. Knowing where these elements are in the genome is critical to our understanding of gene expression, disease and animal health.Aim 1. Identify coding and long noncoding RNA transcripts in the chicken genome.Aim 2. Identify promoter, enhancer, silencer and insulator elements in the chicken genome.Aim 3. Characterize activity states and tissue specificity of cis-regulatory elements.Aim 4. Map distal regulatory elements to their interacting target promoters.
Project Methods
RNA-seq (Aim 1): RNA will be purified using AllPrep DNA/RNA Mini Kit. RNA-seq libraries will be generated using ScriptSeq v2 RNA-Seq Library Preparation Kit to generate whole transcriptome strand-specific libraries. ERCC spike-in controls will be used during library preparation.Libraries will be sequenced with single-end 75 cycles (SE75) on an Illumina Nextseq. Each library will acquire 50M reads for adequate transcriptome coverage. Duplicates from two independent experiments will be performed and correlated for concordance (Pearson's r>0.95).ChIP-seq (Aims 1-3):ChIP-seq will be conducted to annotate promoters (H3K4me3, H3K27me3, H3K27ac), enhancers (H3K4me1, H3K27ac), insulators (CTCF), and to confirm novel transcript annotations (H3K36me3). Tissue will be ground in a liquid nitrogen mortar and pestle and cross-linked for 10 min while thawing in 1% formaldehyde buffer. Chromatin preparation and ChIP-seq will be conducted as we have previously described, using 20 μg of chromatin with 3 μg of antibody.Libraries will be prepared using standard protocols using Illumina Y-adapters. Libraries will sequenced using SE75 cycles on NextSeq for 20M reads per ChIP and input library. As control, a library is constructed for each input chromatin.ATAC-seq (Aim2):ATAC-seq will be performed on ~50,000 cells in replicate as previously described. To increase usable reads, digitonin will be used to reduce mitochondrial DNA from ~50% to ~11% of library reads. We will acquire 50M PE75 reads on a Nextseq. PE reads are necessary to distinguish nucleosomal reads from TF binding sites by filtering for fragment length.Whole genome bisulfite sequencing (Aim 2):WGBS will be performed as previously described using 500 ng of sonicated genomic DNA per library. Lambda DNA is used as a control for determine bisulfite conversion efficiency and spiked in at 0.5%. DNA was end repaired, dA-tailed, and ligated using Illumina compatible, methylated barcoded adapters. Bisulfite treatment is performed and DNA is amplified for 4-8 cycles. Libraries will be sequenced on HiSeq4000 using PE150 cycles for ~10X coverage per replicate after mapping at a ~70% mapping rate (~120M reads). All experiments are performed in replicate, resulting in 20X coverage per methylome.PLAC-seq (Aim 4):Proximity ligation assisted ChIP-seq will be performed using H3K4me3 as recently described. Tissue will be processed as for ChIP-seq. Following isolation of nuclei, a standard HiC protocol is followed through ligation of ends. H3K4me3 was selected to enrich for promoter regions and to map their interacting distal elements. Libraries will be sequenced using PE75 on a NextSeq to a depth of 200M reads each. All experiments will be performed in replicate.Data Analysis.For all methods the quality of the reads will be checked with FastQC tools, and trimmed for low quality (q score < 30) and adapter sequences, followed by removal of duplicates. All reads for this project will be mapped to the chicken genome galGal4 build from UCSC (1.23Gb size).RNA-seq quantification and novel annotations (Aim 1):RNA-seq reads will be aligned using TopHat2. Transcript quantification will be performed by Cufflinks. ERCC spike-in controls will be used to accurately compare expression levels and dynamic ranges across samples. Transcripts will be cross-validated by their presence in other tissue RNA-seq datasets and by the presence of H3K36me3 ChIP-seq signal and mCG from the same tissue.ChIP-seq (Aim 1-3):Following QC, ChIP-seq reads will be aligned using Bowtie2. Replicate reproducibility will be assessed in several ways. We will use IDR for CTCF data. For histone modifications we will perform a bam file correlation as a first pass (r>0.90). Highly correlated duplicates will be merged for subsequent analyses. Peaks are called on individual and merged duplicates using MACSv1.4. ChIP-seq signals will be normalized by subtraction of input using deepTools v2.3 suite.ATAC-seq (Aim 2):Following QC, ATAC-seq reads will be aligned using Bowtie2. Fragments greater than 200bp are filtered as nucleosomal reads. Regions of enrichment will be called using MACS2, narrow peaks. Replicates will be merged for additional analysis.WGBS (Aim 2):WGBS reads are aligned and methylation quantified using both BSeeker2 and Bowtie2using the end-to-end mode. Unmethylated and low methylated regions (UMRs and LMRs) will be identified using MethylSeekRwith FDR < 5% for regions with < 10% (UMRs) or < 50% (LMRs) mCG/CG and > 5 CGs per region. CpG islands (CGIs) coordinates will be obtained for the chicken genome form UCSC (21,664 sites) for analysis.Identification of silencer elements (Aim 2):Per tissue/cell type, starting with ATAC-seq peaks, all annotated TSS are removed. Followed by removal of CTCF, H3K4me3 and H3K4me1 peaks. The remaining collection of ATAC-seq peaks serves as the putative silencer test set. These sites will be subjected to analysis using gkm-SVM model trained on human and mouse silencers. We will intersect our silencer data with mCG levels to determine factors that bind methylated vs unmethylated (LMRs) silencer elements.Characterize activity states of cis-regulatory elements (Aim 3):To classify promoters and enhancers for various activity states, such as active and poised, a two-pronged approach will be taken. First, promoter and enhancer states will be assessed by chromatin peak overlap, requiring at least 50% of the defining signature (H3K4me3 or H3K4me1) overlap an H3K27 modification using bedtools/2.24.0 suite [85]. We will annotate promoters: asactive(H3K4me3+H3K27ac),poised(H3K4me3-only),bivalent(H3K4me3+H3K27me3), orrepressed(H3K27me3 only, or DNA methylation - here H3K4me3 is lacking so annotation as a known TSS or H3K4me3+ATAC-seq peak or UMR in another tissue will be required). Finally, methylation levels will be assessed across all classifications. Similarly, enhancers will be defined asactive(H3K4me1+H3K27ac),poised I(H3K4me1-only),poised II(H3K4me1+H3K27me3). To define binding sites with broad chromatin peaks (on average 1.5kb), we will intersect all classifications with ATAC-seq and UMR/LMR data to define TFBS as promoters or enhancers and of what classification. ATAC-seq peaks and/or LMRs/UMRs with no chromatin signature or silencer assignment will be classified as unknown regulatory elements. Second, we will take an independent classification approach by employing ChromHMM. All histone modification data, DNA methylation data, and ATAC-seq data are input without peak calls to partition or segment the genome into distinct epigenetic states.Identification of tissue/cell-specific genes and regulatory elements (Aim 1 & 3): Since we will generate RNA-seq for 20 tissue and cell types, we will not generate all pairwise comparisons for differential expression, but instead will define tissue- and cell-specific expression. Tissue/cell-specific expression will be determined using an approach in which Shannon entropy is used to rank genes from having equal expression across all groups to having significant expression in one group only. Low values of Qg t indicate tissue specific expression. We use a cutoff of Qg t < 1.5 to define cell-specific expression. Because this method requires read counts as input, we will apply this to identify tissue/cell-specific peak calls from ChIP-seq and ATAC-seq data as we have previously shown for histone data.PLAC-seq (Aim 4):PLAC-seq will be processed using the Ren lab pipeline (https://github.com/r3fang/PLACseq). Interaction sites with H3K4me3 peaks from ChIP-seq data (anchor points) will be filtered against ChIP-seq peaks (H3K4me3, H3K4me1 & H3K27ac), ATAC-seq peaks, and UMRs/LMRs as interactions with valid distal regulatory elements or other promoters.

Progress 03/01/23 to 02/29/24

Outputs
Target Audience: Poultry researchers, Genetics researchers, FAANG community, Poultry industry representatives Changes/Problems:No major problems any longer, but the Hi-C assay requires too many cells which has been an issue for the cell based experiments. We are addressing this by changing the methods. The advantage of all the optimization we have done is that it will provide not only high quality data, but also provide other researchers in the FAANG community updated methods, not just for poultry, but also other species. What opportunities for training and professional development has the project provided?Postdoctoral fellow Theros Ng has learned new techniques and data analysis, leading to obtaining a faculty position as Assistant Professor at Western University, where he will continue to work on poultry science projects involving the sequencing techniques and data analyis acquired through this project. Several research scientiest and postdoctoral fellows at the University of Washington have found positions in industry and other laboratories. Undergraduate and graduate students have been involved in various experiments and data analysis, which will advance their further careers. Graduate student Brandi Sparling finished her PhD and is currently contemplating her own postdoctoral fellowship involving bioinformatic analysis, which she had no experience in previously. Overall, all personell involved has profited from the involvement by learning cutting edge techniques, data analyis, presentation of results both at national and international conferences, networking and career opportunities. How have the results been disseminated to communities of interest?Conferences, workshops, multi-state meetings. Research Day at CVM WU Manuscripts are in preparation Zymo Webinar online What do you plan to do during the next reporting period to accomplish the goals?Finish last assay (Hi-C), by incorporating CUT/TAG method. Initial results seem promising. Complete data analysis and manuscripts

Impacts
What was accomplished under these goals? We have completed 4 out of 5 assays for all cells and tissues proposed with optimized assays and high quality data.Hi-C is stillbeing optimized as the cell number for the assay is very high and the assay as published did not yield data as expected.Weare building on our experience using CUT and TAG as basis for most assays which has yielded greater quality data using less cells. As wewrap up data production for our USDA funded project, we are currently preparing several manuscripts based on the data we have been generating. There is currently a call for papers for a Functional Annotation of Animal Genomes (FAANG consortium) special issue in the journalGenomics:https://www.sciencedirect.com/journal/genomics/about/call-for-papers.We anticipate submitting two manuscripts currently in preparation. A high resolution atlas of gene expression in chicken Andressa O. de Lima Theros T Ng, Brandi Sparling, Kenneth Lai, Yvonne Drechsler, R.DavidHawkins conTAC-seq: An optimized assay for transposase-accessible chromatin in tissues. Eryn Weston, Wendy Olson, Andressa O. de Lima,Alex Isner,Theros Ng,Wellison Diniz, Carl R. Dahlen, Yvonne Drechsler, R.DavidHawkins This will be followed by a more comprehensive epigenome annotation of cis-regulatory elements A compendium of chicken DNA regulatory elements Andressa O. de Lima, Theros T Ng, Eryn Weston,Giovani Perez, Siddarth Gurajala, Brandi Sparling, Jessica Phung, Lisa Griggs, Yvonne Drechsler R.DavidHawkins The work done has been extensive and it is not possible within the constraints of the format to demonstrate all the work and results, so a separate email will be sent to the program leader with details and figures. We are confident that we will provide a high quality resource of data and optimized assays specifically for working with chickens for many poultry researchers in the next few months via our planned publications. 2/20/2024 Organism:Gallus gallus Tissues:20 tissues Total Samples:2 replicates by tissue (40 samples) Sequencing run:PE sequencing Data information: Samples replicate of 20 tissues (40 samples in total), including: Immune Tissues and Cells: Bursa, B cells, T cells, Macrophage differentiation at day 0, day 3, and day6 monocyte, thymus, macrophage from lung, spleen, and kidney) Reproductive Tissues: Ovary, Magnum (od1), Isthmus (od2), and Shell Gland (od3) Muscle Tissues: Thigh muscle (Iliotibialis major) and Breast muscle (Pectoralis major) Intestinal Tissues: Cecum, Ileum, Jejunum RNA sequencing, WGBS, ATAC seq and ChIP seq have all been completed for all samples and tissues.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Yvonne Drechsler, David Hawkins, Theros Ng, Andressa Oliveira De Lima: Genome-Wide Annotation of Cis-Regulatory Elements in Chicken Genome. A1201 PD meeting at PAG31, January 12th, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Hawkins, R. David: Functional Annotation of the Chicken Genome. FAANG workshop at PAG31, January 15th, 2024
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Annual Meeting NE-1834 Technical Committee. Genetics Bases for the Resistance and Immunity to Avian Diseases, Pacific Grove, CA. Sept 21st to Sept 23rd, 2023 Title: Western University of Health Sciences College of Veterinary Medicine Station Report Authors: Yvonne Drechsler, Theros Ng, Brandi Sparling


Progress 03/01/22 to 02/28/23

Outputs
Target Audience:Poultryresearchers,Geneticsresearchers,FAANGcommunity Changes/Problems:The main issue currently is getting sufficient nuclei from the organ macrophages. Despite repeating the assays several times, these cells have not yielded enough RNA, or DNA or nuclei to get good quality data. The PDs are discussing to change some of the proposed organ macrophages and profile other valuable tissues important to poultry research to move the final phase forward. What opportunities for training and professional development has the project provided?Graduate student Brandi Sparling, and postdoctoral associates Theros Ng and Andressa d'Oliviera have all presented at national/international meetings as described in products. Both the PD and Co-PD presented at the Plant and Animal Genome meeting (PAG) in the FAANG and poultry workshop respectively. In addition, collaborators Fiona McCarthy (University of Arizona) and Wesley Warren (University of Missouri) will be presenting at the PAG meeting using resources/data provided by our project. Further collaborations with Dr. McCarthy, Dr. Warren and Dr. Kolteson new projects are developing due to the data generated and interactions initiated at meetings after presentations. How have the results been disseminated to communities of interest?Presentations listed under poducts at various meetings: USDA multistate meetings, AIRG, PAG What do you plan to do during the next reporting period to accomplish the goals?Finish assays, complete data analysisand prepare manuscripts. We are currently generating ChIP-seq data in the remaining 9 cell and tissue types. We are determining differential peaks in key comparisons to determine how usage of regulatory elements correspond to changes in gene expression. Our optimization of ChIP-seq in tissues has improved our ability to generate higher quality data using ATAC-seq to map open chromatin regions. We are currently generating ATAC-seq in biological replicates from the 20 cells and tissue types. Lastly, we have begun optimizing HiChIP to map interactions between regulatory elements. The ATAC-seq and HiChIP experiments will be finished in the next 12 months. Timetable for completion of project: Assay Task Projected Completion RNA Re-extract 3 samples, QC, Analysis April 2023 WGBS Re-sequence one sample, analysis Integration with gene expression data of all samples July 2023 ATAC seq Use CUT/Tag to improve sequencing quality established on CHIP seq samples Analyze, integrate with other data April 2023 January 2024 CHIP seq Apply optimized cut/tag to finish remaining samples, data analysis July 2023 HiCHIP Ongoing optimization Data analysis July 2023 January 2024

Impacts
What was accomplished under these goals? We have further sequenced samples for several assays and are in data analysis/figure production stages for a large part of them. A few samples need to be repeated due to quality control issues either due to inconsistency clustering, low nuclei recovery (particularly on organ immune cells) and some other technical issues. The last assay proposed is in optimization stage and is expected to be completed next year. The vast majority of samples though is in late phases of analysis as follows: Whole Transcriptome. We have generated whole transcriptome profiling of 20 tissues and cell types from two biological replicates (40 samples in total). The Quality Control (QC) of the paired-end (PE) RNA-seq (150bp) was evaluated by FASTQ. On average, we had 50.2 M reads usable per sample (Table 1) after the removed the low-quality reads (Phred score lower than Q24). The trimmed reads were aligned against the reference chicken genome (NCBI Genome version: bGalGal1.mat.broiler.GRCg7b). The total reads aligned ranged from 28.4 M to 348.1M with an average mapping rate of 90.61 % and ranged from 77.89 % to 96.84%. Isoform Characterization. Using StringTie, we identified 116,596 transcript isoforms with a median length of 4,221 bp and a median of 9 exons. Of these, 95,189 (86,3%) are protein-coding, and 14,520 (13,2%) are ncRNAs. A total of 73,772 (63%) are described in the NCBI database, while 5,121 transcripts (4%) have no known previously annotated gene. We predicted 5,486 lncRNAs among all transcripts using FEELnc. Among them, 1,749 are not currently unannotated in the NCBI genome build.The transcript isoforms expressed ranged from 44,109 to 73,554, with a mean of 52,875 transcripts per tissue. Variant calling and Allele-specific expression (ASE). We identified 6,947,161 variants in transcripts across tissues and cells. To date, the ASE analysis has been performed using the intestinal tissues (cecum, ileum, jejunum). We adopted 1,166,529 variants for cecum, 1,947,672 for ileum, and 1,106,045 for jejunum. We identified 6713, 6662, and 7474 significant ASE SNPs (FDR ≤ 0.05) in jejunum, ileum, and cecum, respectively. Among them, 1030 ASE SNPs were identified in common across the intestinal tissues. These analyses are ongoing in the remaining tissues. Whole Genome DNA Methylation. We have completed whole genome bisulfite sequencing (WGBS) on 19/20 tissues and cell types using biological replicates. The final sample is currently being generated. The QC of the PE reads (150pb) sequencing was evaluated by FASTQ. The mapping rate ranged from 71% to 90% per library. The global percent of CG methylation levels ranged from 51% to 67 % per library . Each replicate for each tissue has 17X global coverage on average, with a minimum coverage of 11X and a maximum of 26X. We performed the PCA analysis and the Pearson correlation coefficient. We are currently determining features of DNA methylation that correspond to candidate cis-regulatory elements, such as unmethylated regions as promoters and low methylated regions as enhancers. In addition, we are determining differentially methylated regions in key pairwise comparisons and integrating these regions with changes in gene expression. Histone modification ChIP-seq to annotate candidate cis-regulatory elements. Five histone modifications (H3K27me3, H3K27ac, H3K4me3, H3K4me1, H3K9me3) have been evaluated 11 tissues in biological replicates: Immune tissues (Bursa, thymus); Reproductive tissues (Ovary, shell gland, isthmus, magnum), Muscle Tissues (breast meat (Pectoralis major) and thigh muscle (Iliotibialis major); and Intestinal tissues (Cecum, Ileum, Jejunum). To date, 7 of 11 have been fully processed and described here within. On average, we received 7.70M paired-end reads (PE read, 75bp) per replicate. After QC control using FASTQC, we performed the alignment against the chicken reference genome using the Bowtie2. On average, the replicates showed a mapping rate of 92.94%. The histone modifications pattern was evaluated on UCSC Genome Browser using bigwig files converted by deepTools. For example, the pattern of histone modification H3K27ac, which marks active enhancers and promoters, for the seven completed tissues (bursa, ileum, jejunum, ovary, cecum, thymus, white meat) demonstrates shared and tissue-specific regulatory regions. The peak calling was performed using the SEACR v.13. For the histone modification, H3K27me3, H3K27ac, H3K4me3, H3K4me1, and H3K9me3 total average peaks are 31011, 27678, 25538, 31029, and 29752, respectively. As a QC metric, we determine the Fraction of Reads in Peaks (FRiP) score as a measure of signal to noise. The following average FRiP scores were determined for each histone modification H3K27me3 (0.73), H3K27ac (0.57), H3K4me3 (0.71), H3K4me1 (0.62), H3K9me3 (0.71) across bursa, ileum, jejunum, ovary, cecum, thymus, and white meat tissues. Biological replicates show good correlation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: NE-1834 Genetic Basis for Disease Resistance to Avian Disease Annual Meeting, Newark, DE. Sept 23-24, 2022: Western University of Health Sciences Station Report. Brandi Sparling, Theros T. Ng, Yvonne Drechsler
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: XVI Avian Immunology Research Group Meeting, Newark, DE. Sept 25-28th, 2022. Improving the cluster homolog of immunoglublin-like receptor annotation and the implications of differential expression in innate immune response in the chicken. Brandi Sparling, Theros T. Ng, Yvonne Drechsler
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poultry Science Association Annaual Meeting, San Antonio, TX, July 11-14, 2022. An Update on Transcriptomes of an Array of Chicken Ovary, Intestinal, and Immune Cells and Tissues. Theros T. Ng, R. David Hawkins and Yvonne Drechsler
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Plant and Animal Genome PAG30 meeting Jan 13-18, 2023: Transcriptome characterization of the chicken (Gallus gallus domesticus) genome. Andressa Oliveira de Lima, Theros T. Ng, Lisa M. Griggs, Yvonne Drechsler, R. David Hawkins
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Plant and Animal Genome PAG30 meeting, FAANG workshop Jan 12, 2023: Chicken FAANG Update. R. David Hawkins and Yvonne Drechsler
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Plant and Animal Genome PAG30 meeting, Poultry 1 workshop Jan 13, 2023: Ig-like Receptors in the Chicken: Annotation, Cell and Tissue Distribution, and Their Role in Disease Resistance. Yvonne Drechsler
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Plant and Animal Genome PAG30 meeting, Poultry 2 workshop Jan 14, 2023: Progress toward an immune cell atlas in the chicken. Wesley C. Warren, Edward Ricemeyer, Ashley Meyer, Elaina Sculley, Christine G Elsik1, Susan J. Lamont, Yvonne Drechsler, Melissa S. Monson, Cari J. Hearn, Hans H. Cheng.


Progress 03/01/21 to 02/28/22

Outputs
Target Audience:Poultry researchers, Genetics researchers, FAANG comunity Changes/Problems:The main issue for the last two years have been the delays due tothe Covid pandemic. It affected everything from tissue collection and processing to data generation (sequencing) and subsequently analysis. Both WesternU and UW are in counties that had stringent restrictions for quite some time. It has resolved for now, but it definitely affected the progress of the work. What opportunities for training and professional development has the project provided?Both Dr. Hawkins and I have trained our postdocs in tissue collection, processing and bioinformatic analysis. The postdoc at WU has an animal science background and has expanded his skills in bioinformatics, including RNA sequencing and ATAC sequencing analysis. In addition, he has used this project to apply to AAI for the intersectional fellowship that he was awarded, providing further training in bioinformatics. The postdoctoral researchers at UW Seattle have a background in bioinformatics and have expanded their knowledge to chicken genomics and annotation. In addition, a graduate student at the WesternU laboratory has been trained in tissue processing and bioinformatic analysis, which enabled her to successfully apply for the Poultry Science Cobb Vantress scholarship. How have the results been disseminated to communities of interest?Publication in Frontiers Genetics Transcriptomes of an Array of Chicken Ovary, Intestinal, and Immune Cells and Tissues E. G. Overbey, T. T. Ng, P. Catini, L. M. Griggs, P. Stewart, S. Tkalcic, et al. Frontiers in Genetics 2021 Vol. 12 DOI: 10.3389/fgene.2021.664424 and presentations at the NE1834 meeting at the University of Maryland and PAG workshopes (poultry and FAANG) What do you plan to do during the next reporting period to accomplish the goals?In the following year, we will continue to complete the data generation and analysis. While we have experienced a number of delays due to the COVID-19 pandemic, we are aiming to complete all data production for Aims 1 to 3 in the next 6 months. Soon we will begin data generation for Aim 4 to map interactions betweencis-reglatory elements, for example promoter-enhancer interactions, using Hi-C based methods. This will continue through the next funding period. Data analysis for all aims is still ongoing and will continue through the end of the funding period. We anticipate that the valuable resource being generated will provide the basis for ongoing data analysis studies by our groups and others beyond the funding period.

Impacts
What was accomplished under these goals? We have worked toward our goal of generating transcriptome (RNA-seq) and genome annotation data (ChIP-seq, ATAC-seq and whole genome bisulfite sequencing (WGBS)) from 20 tissues. In our efforts to identify coding and long non-coding transcripts in the chicken genome (Aim 1), we recently published our finding for the majority of tissues and have since continued to finalize RNA-seq and analysis. To date, we have determined that ~30% of transcripts identified are novel compared to NCBI annotations. The vast majority of these are novel isoforms (splice variants) of known genes. However, 5% are completely unannotated genes in the chicken genome. Using computational approaches we have attempted to predict if these unannotated genes were coding or non-coding. We have found that approximately 1 in 7 is predicted to be coding, with the remainder largely predicted to be long non-coding RNAs. These results provide new insight on the chicken transcriptome. One the greatest challenges within the FAANG consortium has been the optimization of ATAC-seq and ChIP-seq on animal tissues to annotatecis-regulatory elements (Aim 2). We have recently made additional progress, by continuing to improve tissue dissociation for each assay and implementing a version of ChIP-seq called CUT&Tag, which leverage the Tn5 transposase used in ATAC-seq, and performs the chromatin immunoprecipitation in situ using intact nuclei. We have now generated high quality data for H3K4me3, H3K4me1, H3K27ac, H3K27me3 and CTCF for 5 of 20 tissues. With these optimizations, we expect data production to move forward rapidly for both ATAC-seq, where we have generated data for nearly half of the proposed tissues, and ChIP-seq, resulting in annotation of hundreds of thousands ofcis-regulatory elements. Our very preliminary analysis of these data are already demonstrating tissue-specific activity of elements (Aim 3).Additional annotation ofcis-regulatory elements comes from WGBS. To date we have generated data for 18 of 20 tissues. Analyses are ongoing to determine unmethylated and low-methylated regions that correspond tocis-regulatory elements. We have also begun to determine differentially methylated regions indicated of the silencing of elements (Aim 3).

Publications

  • Type: Journal Articles Status: Published Year Published: 2021 Citation: AUTHOR=Overbey Eliah G., Ng Theros T., Catini Pietro, Griggs Lisa M., Stewart Paul, Tkalcic Suzana, Hawkins R. David, Drechsler Yvonne TITLE=Transcriptomes of an Array of Chicken Ovary, Intestinal, and Immune Cells and Tissues JOURNAL=Frontiers in Genetics VOLUME=12 YEAR=2021 URL=https://www.frontiersin.org/article/10.3389/fgene.2021.664424 DOI=10.3389/fgene.2021.664424
  • Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: NE1834 Genetic Bases for Resistance and Immunity to Avian Diseases: October 2021; University of Maryland: Transcriptomes of an Array of Chicken Ovary, Intestinal, and Immune Cells and Tissues, presented by Theros Ng (postdoctoral researcher)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: PAGXXIX Poultry Workshop, January 8th, 2021 (Virtual). Transcriptomes of an Array of Chicken Ovary, Intestinal, and Immune Cells and Tissues, presented by Theros Ng (postdoctoral researcher)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: PAGXXIX FAANG workshop February 18th: Update on Chicken FAANG project. Presented by David Hawkins
  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Poultry Workshop, Plant and Animal Genome XXIX Meeting, January 8-12, 2022 Developing Resources for Chicken ncRNAs. Fiona McCarthy, Amanda Cookset, Yvonne Drechsler


Progress 03/01/20 to 02/28/21

Outputs
Target Audience:Poultry researchers, Genetics researchers, FAANG comunity Changes/Problems:The COVID pandemic has affected our research tremendously, particularly in the first months after March of last year. LA county and therefore the university, went initially into complete lockdown, with technical staff only allowed in to take care of tanimals, but no experiments were allowed initially. While this changed eventually, it led to subsequent issues as we had to follow rules on social distancing, how many people were allowed in the lab and how often they should come in. While we kept working on the project, particulary on data analysis and bioinformatics, we couldn't proceed as fast as we needed to in aspects of data generation in the lab. Particularly a lot of the trouble shooting we were trying to do was delayed, such as the assays that required several days of consistent work. Sample sending and processing to sequencing also was delayed, as the University of Washington also had restrictions on presence of personel on campus, including the sequencing facilty. Overall, we were able to accomplish some progress, but certainly not to the extend we had envisioned before the pandemic. What opportunities for training and professional development has the project provided?Dr. Drechsler has presented her data at the NE meeting in Georgia in 2019, where she recruited a postdoctoral research associate and a PhD student to work on the project. Both, postdoc and graduate student, are originally from the field of poultry science, and are nowalso taking online courses in bioinformatics to increase the capabilities for data analysis in the Drechsler lab. Dr. Drechsler is continuing to increase collaborations with different researchers in poulty fields. Dr. Hawkins has had undergraduate students trained on epigenomic assays in his laboratory aspart of this project, as well as postdocs and technical staff. A new bioinformatics postdoctoral fellow was recruited from the agriculture field with a background in bovine genetics and transcriptomics, and will start later this year. How have the results been disseminated to communities of interest?PAG was canceled, as well as several other conferences. Dr. Drechsler was able to present at the NE-1834 multistate group in September of 2020. A publication was submitted in February of 2021 to Frontiers Genetics and is under review. What do you plan to do during the next reporting period to accomplish the goals?FInish analysis on transcriptome and publish WGBS: The remaining sample replicates are either awaiting quality control sequencing or waiting to be furthersequenced to the desired depth. WGBS will be completed in the next 6 months. Finish ATAC seq Get ChIP seq to final QC stage and start PLAC seq/Hi C, depending on sample size. The Hawkins lab is currently establishing procedures, particularly on ChiP seq, that require less cells. This is especially helpful when extracting tissue macrophages, as these have been low in numbers. In situ chromatin precipitation is being optimized for tissues to compensate for data qualtiy and tissue dissociation troubleshooting.

Impacts
What was accomplished under these goals? RNA libraries are completed and submitted for publication at Frontiers Genetics. We were able to extract tissue macrophages and T cells that we had not been able to obtain the previous year. We are still working to polarize T cells into Th1/Th2 and are currently testing samples by PCR. Whole genome bisulfite sequencing (WGBS) has been completed to detect DNA methylation changes genome-wide for 10 of 20 proposed samples in replicate. ATAC seq libraries are in progress, we had some QC failures with previously runcells and tissues, but are confident we will finish those this year, pending also the tissue extracted cells and polarized T cells.We are considering to analyze CD25 T cells as alternative, if we can't polarize the T cells. ChiP seq libraries have been sequenced and are being re optimized for most tissues and cells. It is necessary to include more sequencing for optimization because other quality indicators have not been sufficient. Repeated optimization and sequencing has yielded higher quality data and will be continued.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Transcriptomes of an array of chicken ovary, intestinal, and immune cells and tissues. Submitted February 5th 2021. Frontiers Genetics Special Issue: Livestock Genomics
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2020 Citation: NE-1834 2020 Virtual Meeting: Functional Annotation of the Chicken Genome Update


Progress 03/01/19 to 02/29/20

Outputs
Target Audience:Poultry researchers, Genetics researchers, FAANG community (Fuctional Annotation of Animal Genomes). Changes/Problems:Optimization of assays required more steps including more sequencing, but is yielding results. If we can't successfully polarize T cells this year, we will reconsider to profile regulatory T cells instead. This was also discussed with Wes Warren who is currently profiling immune cells via single-seq RNA assays. What opportunities for training and professional development has the project provided?Dr. Drechsler has gained more knowledge in bioinformatics and epigenetics resulting in several addtional collaborations. She has attended bioinformatics training workshops. Several undergraduate, graduate and veterinary students have had opportunities in both Dr. Drechsler's and Dr. Hawkins' labs to learn cutting edge techniques and be exposed to the field of bioinformatics. How have the results been disseminated to communities of interest?Currently, most dissemination has happened via presentations to various poultry multi state groups such as NE-1834, NC-1170, etc. What do you plan to do during the next reporting period to accomplish the goals?Continue with sample collection of tissue macrophages and T cells. Continued optimization of all assays and sequencing. Publication is targeted for the end of the year.

Impacts
What was accomplished under these goals? We have several assays in various stages: RNA libraries are near completion with exception of tissue macrophages that required optimization of cell isolation, as well as Th1/Th2 cells that are not polarized like mammalian T cells. We are still working on the generation of these polarized cells. DNA libraries are near completion with exception of Th1/Th2 cells. ATAC seq libraries are in progress and completed for most cells and tissues, apart from the Th polarized cells and tissue macrophages. We will finish these in the near future. ChiP seq libraries have been sequenced and are being re optimized for most tissues and cells. It is necessary to include more sequencing for optimization because other quality indicators have not been sufficient. Repeated optimization and sequencing has yielded higher quality data and will be continued.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: 2019: NE-1834: Yvonne Drechsler: Functional Annotation of the Chicken Genome 2020: PAG XXVII FAANG Workshop: David Hawkins, Yvonne Drechsler et al.: Epigenomic Annotation of Candidate cis-Regulatory Elements in the Chicken Genome. 2020 PAG XVII: Poultry Workshop: Yvonne Drechsler, David Hawkins, Suzana Tkalcic: Infectious Bronchitis Virus Infection Affects Chromatin Accessibility and RNA Differential Expression in a Tissue-Specific Manner. with update on FAANG project


Progress 03/01/18 to 02/28/19

Outputs
Target Audience:Poultry researchers, Genetics researchers, FAANG community (Fuctional Annotation of Animal Genomes). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Professional development: Increase in expertise for epigenetic assays particularly in chicken cells and tissues. Attendance of workshops with presentations. Attendance and presentation at USDA multistate group meeting such as NE-1334. Training of veterinary and graduate students in research. How have the results been disseminated to communities of interest?See conference previously listed. What do you plan to do during the next reporting period to accomplish the goals?Finish all sample collection, assays and sequencing. This will be followed by more in depth bioinformatic analysis. Publication of results.

Impacts
What was accomplished under these goals? We have collected samples from the ADOL 6x7 F1 chickens sent to us from Hans Cheng. These include macrophages, B cells, T cells, and reproductive organs according to revisions requested by USDA for Year 1 and Year 2 funding only. We are working on polarizing the T cells currently and extracting lung macrophages, but will be getting more chicks in spring.We have extracted RNA and done quality control from most samples including initial bioinformatics and are waiting for the T cell populations before RNA sequencing (Aim 1). DNA has been isolated, undergone quality control and is ready to be sequenced in the next weeks (Aim 3 and 4). Other assays such as ChiP seq and ATAC seq are ongoing/being optimized for the individual cells and tissues. (Aim 2-4).

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: PAG 2019 "Functional Annotations of Animal Genomes (FAANG) Workshop": Epigenomic Landscapes from Various Cells and Tissues of Gallus Gallus. Yvonne Drechsler, David Hawkins and Suzana Tkalcic. PAG 2019 "Poultry I workshop": Epigenetic Mapping of the Chicken Genome: Implications for Disease Resistance and Production. Yvonne Drechsler, David Hawkins and Suzana Tkalcic.