Progress 01/01/24 to 12/31/24
Outputs
Target Audience:Animal genome community and livestock industry Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Current work has provided research opportunities to several undergraduate students, three graduate students, 2 postdocs, and project scientist at UC Davis. 1 Ph.D. student, 1 master student and 2 undergraduate students have been trained to isolate high quality RNA and construct RNA libraries, and further bioinformatic analysis How have the results been disseminated to communities of interest?Research results have been presented in Plant and Animal Genome meeting, Poultry Science Association annual meeting What do you plan to do during the next reporting period to accomplish the goals?We are planning to conduct the following activities: 1. Finalize the SNP Reference Panel and Perform Imputation: · Complete construction and validation of the SNP reference panel by integrating high-quality variants from F0 and F18/F19 AIL populations. · Perform genotype imputation across the F34 cohort using this panel to improve SNP density and accuracy for GWAS. 2. Conduct Genome-Wide Association Studies (GWAS): · Utilize imputed SNPs and curated phenotypic data to perform GWAS for key growth-related traits. · Identify and validate QTL regions contributing to phenotypic variation. 3. Long-Read RNA-seq Expansion and Analysis: · Complete long-read sequencing of remaining jejunum samples, estimate finish by the end of Spring 2024. · Continue isoform-level transcriptome assembly and perform differential transcript usage analysis. · Quantify transcript-level expression and perform eQTL and sQTL analyses integrating genotype and transcriptomic data. 4. Multi-Omics Integration: · Integrate genomic, transcriptomic, metabolomic, and phenotypic data using co-expression networks and statistical modeling. · Identify regulatory variants, isoforms, and metabolites linked to complex growth traits. 5. Manuscript Preparation and Public Data Release: · Prepare multiple manuscripts focused on GWAS, eQTL/sQTL, novel isoforms, and multi-omics integration. · Submit relevant sequencing and metadata to public repositories and GitHub to ensure reproducibility and community access.
Impacts
What was accomplished under these goals?
Phenotype and Sample Collection: All 376 F34 advanced intercross chickens tissue samples were collected and phenotypic analysis were conducted at day 19 and day 33. Following histological assessments conducted in 2023, additional phenotypic analysis was performed on the remaining tissue samples, including quantification of growth-related traits and data curation for downstream genetic analyses. The complete phenotypic dataset were finalized and subjected to quality control. To complement transcriptomic and genomic data, we conducted untargeted metabolomics profiling of liver samples using gas chromatography-time of flight mass spectrometry (GC-TOF MS). The analysis focused on primary metabolic pathways, including amino acid, carbohydrate, lipid, and nucleotide metabolism. Metabolite data will be integrated with phenotypic measurements and sequencing data to investigate molecular signatures associated with divergent growth rates. Quality control and normalization of metabolite data were completed, and differential abundance analyses are underway to identify metabolites associated with high and low growth performance. Whole Genome Sequencing and GWAS: Re-sequenced samples with low initial coverage (<0.5X) were successfully processed, increasing the overall average genome coverage to ~1X across the F34 cohort. Single nucleotide polymorphisms (SNPs) were called using the GATK best practices workflow, followed by multi-step filtering to ensure high-confidence variant sets with the following parameters: GATK joint-called SNPs: 3,955,169.Post-VariantFiltration: 3,829,040. Biallelic SNPs retained: 3,742,356. MAF ≥ 0.005 SNPs: 2,624,575. To improve imputation accuracy for the low-pass WGS data of the F34 chickens --which is critical for GWAS analysis --we are currently constructing a high-resolution SNP reference panel. This panel integrates 30X whole-genome sequencing data from F0 modern broiler (sequenced at the UC Davis Genome Center) and F0 Fayoumi (sourced from a public database), from which a total of 8,311,812 SNPs were called using the GATK pipeline. In addition, we incorporate 600K SNP array data from the F18 and F19 generations of the AIL population (Van Goor et al., BMC Genomics 2016; Van Goor et al., Genet Sel Evol 2015). These generations share a close ancestral relationship with the F34 population, making them well-suited for reference-based imputation. Once completed, this comprehensive reference panel will enable accurate genotype imputation, improve SNP density, and enhance the resolution and power of downstream association analyses. Long-Read RNA Sequencing: After establishing and optimizing the long-read sequencing workflow in 2023, we upgraded the RNA sequencing protocol to utilize the newly released Oxford Nanopore cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24), released in late July 2024, along with the R10.4.1 flow cell. RNA extraction and library preparation protocols were further refined for reproducibility and high-throughput capability. Using this upgraded platform: All 376 liver RNA samples were sequenced, generating 1,020,710,258 full-length mapped reads with an average read length of 1,072 bp. All pectoralis major muscle samples were sequenced, producing 1,656,738,650 raw reads with an average N50 mapped length of 1,208 bp. Jejunum RNA was extracted from all samples. A pilot batch of 48 jejunum samples was sequenced to test the pipeline, yielding 167,804,841 mapped reads with an average N50 mapped length of 931 bp. Initial transcriptome analysis revealed over 830,000 transcript isoforms, averaging 28.5 isoforms per gene. Growth-related genes such as IGF-1, IGF1R, and mTOR exhibited expanded transcript diversity, including novel isoforms not present in the reference annotation. Downsampling and saturation analyses confirmed that ≥2 million mapped reads per sample are required to capture transcriptome complexity comprehensively.
Publications
- Type:
Other Journal Articles
Status:
Published
Year Published:
2024
Citation:
Comparative Genomics and Epigenomics of Transcriptional Regulation. Annual Review of Animal Biosciences
- Type:
Other Journal Articles
Status:
Published
Year Published:
2024
Citation:
Enriched atlas of lncRNA and protein-coding genes for the GRCg7b chicken assembly and its functional annotation across 47 tissues
- Type:
Other Journal Articles
Status:
Published
Year Published:
2024
Citation:
Age-dependent genetic architectures of chicken body weight explored by multidimensional GWAS and molQTL analyses.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2024
Citation:
Harnessing Oxford nanopore sequencing technology to uncover poultry genetics.
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Progress 01/01/23 to 12/31/23
Outputs
Target Audience:Animal genome community and livestock industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Current work has provided research opportunities to several undergraduate students, three graduate students, 2 postdocs, and project scientist at UC Davis. 1 Ph.D. student, 1 master student and 2 undergraduate students have been trained to isolate high quality RNA. How have the results been disseminated to communities of interest?Currently still waiting for the sequencing data for further analysis. What do you plan to do during the next reporting period to accomplish the goals?We plan to conduct a heritability analysis focusing on the growth-related complex traits in the F34 advanced intercross chicken population. Following this, we aim to estimate linkage disequilibrium (LD) across different generations, including F0 modern broiler, F0 Fayoumi, and F34 advanced intercross chicken. Upon receiving re-sequenced data with adequate coverage, we will proceed with a genome-wide association study (GWAS) to identify quantitative trait loci (QTLs). Oxford Nanopore has upgraded their chemistry to the cDNA-PCR Barcoding Kit V14 (SQK-PCB114.24) and PromethION flow cell (R10.4.1), discontinuing the older versions of the library kit and flow cell. To ensure an optimized protocol for our samples, we will acquire the newer versions of the flow cell and kits. Once the protocol meets our expectations, we will proceed to generate long-read RNA sequence data from all liver , muscle and jejunum samples and subsequently perform downstream analysis.
Impacts
What was accomplished under these goals?
Animal Tissue (Phenotype): We conducted histological analysis on liver, pectoralis major breast muscle, jejunum, and ileum samples from both the top 20 and lowest 20 growth rate chicks, resulting in a total of 160 histology samples. All samples underwent H&E staining. Specifically, for jejunum and ileum samples, we assessed various phenotypic parameters, such as basal membrane length, crypt count, villi count, average villi height, average crypt depth, and the villous height to crypt depth (V:C) ratio. Whole genome sequence: Upon receiving all 376 F34 advanced intercross chicken whole genome sequence data from Neogen, we commenced a quality control procedure to assess data integrity. This encompassed several stages, such as adaptor trimming, read trimming and filtering, and PCR duplicate removal. Subsequently, we aligned the sequences to both the GRCg7b and GRCg7w chicken reference genomes. Following alignment, we evaluated the depth and uniformity of sequence coverage across the genome. The collective coverage across all samples averaged 0.77X. Samples exhibiting coverage below 0.5X were subjected to re-sequencing to achieve higher coverage levels. Similarly, we conducted sample quality control on the 30X F0 modern broiler whole-genome sequencing data upon receipt from UC Davis genome center. The F0 Fayoumi whole-genome sequence data was sourced from a public database, and we subjected it to the same preprocessing steps as other samples. Subsequently, we employed GATK best practices workflows to call SNPs. Long-read RNA sequence: We established the Oxford Nanopore long-read sequencing platform (P2 solo device) within the laboratory and conducted various tests to determine the optimal conditions for RNA long-read sequencing using the cDNA-PCR Barcoding Kit V11 (SQK-PCB111.24) and PromethION flow cell (R9.4.1) on liver samples. Our tests focused on identifying the best RNA extraction method, determining the appropriate PCR cycle numbers, and optimizing PCR extension times to refine the sequencing protocol for optimal results. Through these efforts, we identified a total of 20,449 transcripts from approximately 5.9 million mapped full-length reads. Subsequently, a down-sampling analysis was executed, indicating that a minimum of 2 million mapped reads per sample is necessary to detect expressed transcripts.
Publications
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Lingzhao Fang, Dailu Guan, Zhonghao Bai, Xiaoning Zhu, Conghao Zhong, Yali Hou, Houcheng Li, Fangren Lan, Shuqi Diao, Yuelin Yao, Bingru Zhao, Di Zhu, Xiaochang Li, Zhangyuan Pan, Yahui Gao, Yuzhe Wang, Dong Zou, Ruizhen Wang, Tianyi Xu, Congjiao Sun, Hongwei Yin, Jinyan Teng, Zhiting Xu, Qing Lin, Shourong Shi, Dan Shao, Fabien Degalez, Sandrine Lagarrigue, Ying Wang, Ming-Shan Wang, Minsheng Peng, Dominique Rocha, Mathieu Charles, Jacqueline Smith, Kellie Watson, Albert Buitenhuis, Goutam Sahana, Mogens Lund, Wesley Warren, Laurent Frantz, Greger Larson, Susan Lamont, Wei Si, Xin Zhao, Bingjie Li, Haihan Zhang, Chenglong Luo, Dingming Shu, Hao Qu, Wei Luo, Zhenhui Li, Qing-Hua Nie, Xiquan Zhang, Ruidong Xiang, Shuli Liu, Zhe Zhang, Zhang Zhang, George Liu, Hans Cheng, Ning Yang, Xiaoxiang Hu, Huaijun Zhou. The ChickenGTEx atlas: the genetic regulation of multi-tissue and single-cell transcriptome signatures in chickens. BioRxiv
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Progress 01/01/22 to 12/31/22
Outputs
Target Audience:Animal genome community and livestock industry. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Current work has provided research opportunities to several undergraduate students, three graduate students, 2 postdocs, and project scientist at UC Davis. Team of 23 people from multiple labs were involved in sample collection. 1 Ph.D. student, 1 master student and 3 undergraduate students have been trained to isolate high quality DNA. How have the results been disseminated to communities of interest?Currently still waiting for the sequencing data for further analysis. What do you plan to do during the next reporting period to accomplish the goals?The focus for the next reporting period will be analyzing the whole genome sequencing data to identify the genetics variants and perform QTL mapping. In addition, 1128 high quality RNA will be isolated from pectoralis major muscle, liver, and jejunum along with the libraries that capture the full-length transcriptome. These samples will be submitted to UC Davis Genome center for Oxford Nanopore long-read sequencing.
Impacts
What was accomplished under these goals?
A total of 376 chicks in a F34 advanced intercross chicken population from Iowa State University poultry farm were shipped to UC Davis avian facility. Body weights of each bird were measured weekly. A total of 7,896 tissue samples were collected (21 tissues/chicken) on day 19 and day 33 (188 chicken at each time point). The 21 tissues included blood, thymus, pectoralis major muscle, liver, heart, lung, spleen, adipose tissue (abdominal fat), bursa, bone, bone marrow, cortex, cerebellum, hypothalamus, anterior pituitary gland, jejunum, ileum, ileum content, cecum, cecum content, testis. During the necropsy, the weights of pectoralis major breast muscle, pectoralis minor breast muscle, liver, heart, and spleen were recorded. Additionally, pectoralis major breast muscle, liver, and jejunum for further histology assay from a total of 40 birds from the highest and lowest growth rate groups (20 birds each) were collected. Total RNA will be isolated from pectoralis major breast muscle, liver and jejunum and will be sequenced using Nanopore long-read sequencing. Moreover, high quality DNAs were isolated from all 376 F34 advanced intercross chicken and were submitted to Neogen for low-pass whole-genome sequencing. F0 modern broiler DNA was also isolated and submitted to UC Davis genome center to 30x whole-genome sequencing.
Publications
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