Progress 12/15/09 to 12/14/13
Outputs
Target Audience: Poultry researchers (academic and industry) and producers, poultry breeders, and avian scientists. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? The project has provided ample training opportunities not only for the post-doctoral fellow and graduate students, but also to a number of undergraduate research volunteers and other graduate students who assisted and learned various protocols. Additionally, students were afforded the opportunity to present their findings at major conferences and prepare their work for publication in scientific outlets. How have the results been disseminated to communities of interest? All the data generated during the reporting period have been disseminated first at relevant scientific meetings as oral and/or poster presentations, in annual technical reports, as well as manuscripts and public databases. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The turkey genome sequence along with the detailed tissue transcriptomes provide resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. RNA-Seq has become a routinely used approach to study gene expression profiles by taking advantage of high-throughput sequencing technologies. This project constitutes the first report on using RNA-Seq to gain insight into the transcriptional profiles associated with tissue development in the domesticated turkey. As all analyses are being completed and results published, this comprehensive overview of gene expression is providing detailed insight into tissue-specific transcriptomic changes during turkey development, information that can be employed in various research and selection aspects. Overall, the turkey genome sequencing project has generated significant genomics resources not only for the turkey, but also for other poultry and vertebrate species for comparative analyses. Data characterizing the initial genome assembly, annotation, and analyses have been published in multiple appropriate outlets and made available to the public through freely-accessed databases, and additional and updated resources will be available soon once all are validated. The integrated sequencing and assembly approach developed in this project has provided a model for both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest, as evidenced by over 100 citations of the original turkey genome paper (over the last 3.5 years). Having a fully sequenced and highly annotated ‘reference’ turkey genome is radically enhancing our ability to explore the avian immune system, study host-pathogen interactions, and better characterize major infectious diseases of economic consequences. Equally important, it is providing a valuable resource for studying genetic variations underlying other economically important traits in poultry, effectively allowing comparative genomic analyses of avian and vertebrate species, and collectively accelerating our ability to positively impact the productivity and sustainability of the US and world poultry industry.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Kim S., Settlage R.E., Reed K.M., and Dalloul R.A. Using RNA-Seq to characterize tissue transcriptomes at various developmental stages of the domestic turkey. Annual meeting of the Poultry Science Association, San Diego, CA. July 22-25, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Dalloul R.A. Next generation sequencing strategies for characterizing the turkey genome. Symposium: Applying Next Generation Sequencing to Solve Poultry Problems; Annual meeting of the Poultry Science Association, San Diego, CA. July 23, 2013.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Reed K.M., Monson M.S., Settlage R.E., Mendoza K.M., and Dalloul R.A. MHC composition and variation in turkeys of the Americas. Annual Meeting of the American Association of Immunologists, Honolulu, HI. May 3-7, 2013.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Dalloul R.A., Zimin A.V., Settlage R.E., Kim S., and Reed K.M. Next generation sequencing strategies for characterizing the turkey genome. Poultry Science 93:479-484. 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kim S., Settlage R.E., Reed K.M., Dalloul R.A. Using RNA-Seq to characterize brain development in the maturing domesticated turkey. Plant and Animal Genome XXII, San Diego, CA. January 11-15, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Kim S., Settlage R.E., Reed K.M., Dalloul R.A. Transcriptome analysis of early reproductive tract development in the turkey using RNA-Seq. Plant and Animal Genome XXII, San Diego, CA. January 11-15, 2014.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Reed K.M. Characterizing the developmental transcriptome of the domestic turkey. Poultry Workshop, Plant and Animal Genome XXII, San Diego, CA. January 11, 2014.
- Type:
Other
Status:
Other
Year Published:
2013
Citation:
Dalloul R.A. Sequencing the turkey genome: challenges and usefulness. Fall Seminar Series, Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA. October 7, 2013.
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Progress 12/15/11 to 12/14/12
Outputs
OUTPUTS: Since releasing the first draft of the turkey genome sequence in 2010, efforts have continued to improve the assembly, gene annotation, and further analyze the genome. The initial assembly build (2.01) represented ~89% of the genome sequence with 17X coverage depth (931 Mb). Sequence contigs were assigned to 30 of the 40 chromosomes with approximately 10% of the assembled sequence corresponding to unassigned chromosomes (Chr_Un). The turkey genome sequence has been refined through both genome-wide and area focused sequencing, including additional shotgun and paired-end sequencing, and targeted sequencing of chromosomal regions with low or incomplete coverage. These subsequent sequencing efforts have improved the sequence assembly resulting in three genome builds (3.0, 4.0, 5.0) since the 2010 release. For example, construction of additional 3Kb and 8Kb libraries with sequencing on Roche/454 Titanium system has improved scaffold and contig sizes and further closed many gaps. The key improvements in 3.0 were an additional 20Mb of sequence scaffold, significantly reduced the number of independent scaffolds, which added to the total length of the chromosome sequences. Also, it produced bigger contigs resulting in 35% improvement in the N50 contig size and higher genome coverage increasing genome redundancy to 25X and overall coverage by roughly 2%. Construction and sequencing of more 3Kb and 8Kb Roche/454 libraries further improved the assembly. Using the original data and incorporating additional 3Kb and 8Kb PE reads, a significantly improved turkey genome assembly was produced with N50 contig size almost tripled (from 12.5Kb to 34.5Kb) and N50 scaffold size going up from 1.5Mb to 2.3Mb. The total amount of sequence in build 4.0 went up from 951Mb to 1,009Mb and overall coverage depth to over 30X. These builds (3.0 and 4.0) were created to assess the value of the newly added sequences and did not undergo extensive annotation as additional sequences are being added. As such, neither build was released as efforts continued to enhance coverage of the smaller microchromosomes and the sex chromosomes, detailed gene annotation, and further genomic analysis of the latest build (5.0) that was recently completed. For this build (UMD 5.0) employed the Illumina GAII for sequencing pools of BAC clones from both the CHORI260 and TAMU libraries with end sequences with BLAST hits to the Z chromosome, ChrUn and an additional BAC pool derived from screening of the CHORI260 library for MHC-related sequences (MGA18). Improvements over build 4.0 include a total sequence of 1,075Mb and more than doubled N50 scaffold size (5.7Mb). Chromosome files are currently being generated and detailed gene annotation is underway. To help with the latter, RNA was isolated from several tissues of female and male turkeys at various developmental stages and transcriptome sequencing is underway using the Illumina Hi-Seq platform. Tissues included in this project were thymus, bursa of Fabricius, spleen, brain, heart, liver, pancreas, proventriculus, gizzard, duodenum, jejunum, ileum, breast and thigh muscles, and various sections of the reproductive organs. PARTICIPANTS: Rami A. Dalloul, Principal Investigator/Project Director; Kent M. Reed, Ed J. Smith, and Otto Folkerts, co-investigators; Robert Settlage, Aleksey Zimin and James York, collaborators. TARGET AUDIENCES: Poultry researchers (academic and industry) and producers, turkey breeders, and avian scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the initial sequence assembly, a synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map was employed. This integrated approach has provided a model for both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest, as evidenced by over 70 citations of the turkey genome paper in 2 years. The turkey genome sequence has afforded detailed comparative analysis of avian genomes particularly between chicken and turkey as outlined in the published work. Furthermore, although some important genes are still missing from the assembly (e.g. MHC class IIα) and several cases of incomplete or inaccurate annotation are known, the current turkey genome sequence has become an important tool in the analysis of transcriptome sequences. Current RNA-Seq projects utilize alignments to both the genome sequence and the annotated turkey and chicken gene sets. Projects investigating immunogenetics and toxicology are focusing on identifying important gene modulators of the immune and the inflammatory response. For example, the turkey genome sequence has provided the framework for identifying undescribed regions of the avian MHC. Other projects focus on identifying key elements of the host immune response to a number of impactful poultry diseases. Transcriptomic profiling of disease-stricken birds (e.g. turkey cellulitis) and those infected with food-borne pathogens (e.g. Salmonella) are currently underway. It is well known that susceptibility and resistance to disease present the most challenging phenotypes to study. This approach will not only answer fundamental questions of host-pathogen interactions, but will also aid in further improving the transcriptional annotation of the turkey genome. Also, the whole genome sequence served as the anchor and coordinating platform for genome-wide SNP discovery and the future development of SNP-chip resources under development (Aslam et al., 2012). The initial sequence identified 920,126 single nucleotide variants of which 601,490 were considered as having strong support, with the most recent whole genome SNP discovery in 32 birds resulted in the detection of 5.49 million putative SNPs compared to the reference genome. One ultimate goal of these projects is to provide the community with robust tools and resources to facilitate immediate and long term applications of genome-wide selection for specific traits including production, disease resistance, and reproduction parameters. The most current sequence covers over 95% of the turkey genome and should yield a much improved gene annotation level once completed in early 2013, making it a valuable resource for comparative genomics and to study genetic variations underlying economically important quantitative traits in poultry.
Publications
- Parra ZE, Mitchell K, Dalloul RA, Miller RD. 2012. A second TCRd locus in Galliformes uses antibody-like V domains: insight into the evolution of TCRd and TCRu genes in tetrapods. The Journal of Immunology 188:3912-3919.
- Dalloul RA, Long JA, Zimin AV, Aslam L, Beal K, Blomberg LA, Bouffard P, Burt DW, Crasta O, Crooijmans RP, Cooper K, Coulombe RA, De S, Delany ME, Dodgson JB, Dong JJ, Evans C, Frederickson KM, Flicek P, Florea L, Folkerts O, Groenen MA, Harkins TT, Herrero J, Hoffmann S, Megens HJ, Jiang A, de Jong P, Kaiser P, Kim H, Kim KW, Kim S, Langenberger D, Lee MK, Lee T, Mane S, Marcais G, Marz M, McElroy AP, Modise T, Nefedov M, Notredame C, Paton IR, Payne WS, Pertea G, Prickett D, Puiu D, Qioa D, Raineri E, Ruffier M, Salzberg SL, Schatz MC, Scheuring C, Schmidt CJ, Schroeder S, Searle SM, Smith EJ, Smith J, Sonstegard TS, Stadler PF, Tafer H, Tu ZJ, Van Tassell CP, Vilella AJ, Williams KP, Yorke JA, Zhang L, Zhang HB, Zhang X, Zhang Y, Reed KM. 2010. Multi-platform Next Generation Sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis. PLoS Biology 8:e1000475.
- Published Abstracts and Presentations: Dalloul RA, Zimin AV, Settlage RE, Reed KM. 2012. The turkey genome sequence: an update on the assembly and latest build. The 33rd Conference of the International Society of Animal Genetics, July 15-20, Cairns, Australia.
- Monson MS, Settlage RE, Mendoza K, Rawal S, Coulombe RA, Dalloul RA, Reed KM. 2012. Utilizing spleen transcriptome analysis to identify responses to aflatoxin B1 in the domestic turkey. The 33rd Conference of the International Society of Animal Genetics, July 15-20, Cairns, Australia.
- Monson MS, Settlage RE, Mendoza KM, Rawal S, Coulombe RA, Dalloul RA, Reed KM. 2012. High throughput RNA-sequencing of the domestic turkey liver transcriptome: Response to aflatoxin B1. The 33rd Conference of the International Society of Animal Genetics, July 15-20, Cairns, Australia.
- Monson MS, Settlage RE, Rawal S, Coulombe RA, Reed KM. 2012. Spleen transcriptome response to aflatoxin in the domestic turkey. Plant and Animal Genome XX, January 13-18, San Diego, CA.
- Kim S, Smith EJ, Reed KM, Miska KB, Dalloul RA. 2012. Evolution of nucleotide-binding oligomerization domain protein 2 (Nod2). Plant and Animal Genome XX, January 13-18, San Diego, CA.
- Reed KM, Dalloul RA. 2011. The turkey genome sequence: implications for enhancing turkey biology. In: Turkey production and Health: An Update. Proceedings of the 6th International Meeting of the Working Group 10 (Turkey) of WPSA (Ed. Hafez, H.M), Berlin. Mensch & Buch Verlag, Germany. ISBN 978-3-86387-094-2. Pp. 23-29.
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Progress 12/15/10 to 12/14/11
Outputs
OUTPUTS: Since the release of the turkey genome sequence in 2010, efforts to improve the assembly, gene annotation, and further genomic analyses continue. The initial assembly build (2.01) represented ~89% genome coverage (931 Mb). Sequence contigs were assigned to 30 of the 40 chromosomes with approximately 10% of the assembled sequence corresponding to unassigned chromosomes (Chr_Un). The genome sequence was constructed from an inbred bird of a Nicholas line and with the continuing USDA funding, we are now working to extend our sequencing of the turkey genome with a goal of 99% coverage. We are refining the turkey genome sequence through both genome-wide and area focused sequencing, including additional shotgun and paired-end sequencing and targeted sequencing of chromosomal regions with low or incomplete coverage. These subsequent sequencing efforts have improved the sequence assembly resulting in two genome builds (3.0 and 4.0) since the 2010 release. For example, construction of additional 3Kb and 8Kb libraries with sequencing on Roche/454 Titanium system has improved scaffold and contig sizes and further closed many gaps. The key improvements in this assembly were an additional 20Mb of sequence scaffold, significantly reduced the number of independent scaffolds, which should add to the total length of the chromosome sequences. Also, it produced bigger contigs resulting in 35% improvement in the N50 contig size and higher genome coverage increasing genome redundancy to 25X and overall coverage by roughly 2%. Construction and sequencing of more 3Kb and 8Kb Roche/454 libraries further improved the assembly. Using the original data and incorporating additional 3Kb and 8Kb paired-end reads produced at Virginia Tech (VBI), a much improved turkey genome assembly was produced and was significantly better than the published one. The N50 contig size almost tripled going from 12.5Kb to 34.5Kb, N50 scaffold size went up from 1.5Mb to 2.3Mb. The total amount of sequence in the assembly (4.0) went up from 935Mb to 1,01Mb and overall coverage depth to 30X. These builds (3.0 and 4.0) were created to assess the value of the newly added sequences and did not undergo extensive annotation as additional sequences are being added. As such, neither build was released as efforts continue to enhance coverage of the smaller microchromosomes and the sex chromosomes, detailed gene annotation, and further genomic analysis of the latest build (5.0) expected to be completed in 2012. Another approach being used is the sequencing of pools of BAC clones. BACs with end sequences assigned to the Z/W chromosomes, as well as those assigned to Chr_Un or not placed in the current build, have been isolated from the two turkey BAC libraries (CHORI-260 and 078-TKN-MI). These pools were deeply sequenced using Illumina GAIIx technology and are currently being incorporated into the latest build resulting in an updated sequence assembly (5.0), which completion and release are anticipated within the coming year. PARTICIPANTS: Rami A. Dalloul, Principal Investigator/Project Director; Kent M. Reed, Ed J. Smith, and Otto Folkerts, co-investigators; Aleksey Zimin and James York, collaborators. TARGET AUDIENCES: Poultry researchers (academic and industry) and producers, turkey breeders, and avian scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
For the initial sequence assembly, a synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map was employed. This integrated approach has provided a model for both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest, as evidenced by over 40 citations of the turkey genome paper in 1.5 years. The turkey genome sequence has afforded detailed comparative analysis of avian genomes particularly between chicken and turkey as outlined in the published work. Furthermore, although some important genes are still missing from the assembly (e.g. MHC class IIα) and several cases of incomplete or inaccurate annotation are known, the current turkey genome sequence has become an important tool in the analysis of transcriptome sequences. Current RNA-Seq projects utilize alignments to both the genome sequence and the annotated turkey reference gene set. Projects investigating immunogenetics and toxicology are focusing on identifying important gene modulators of the immune and the inflammatory response. For example, the turkey genome sequence has provided the framework for identifying undescribed regions of the avian MHC. Other projects focus on identifying key elements of the host immune response to a number of impactful poultry diseases. Transcriptomic profiling of disease-stricken birds (e.g. turkey cellulitis) and those infected with food-borne pathogens (e.g. Salmonella) are currently underway. It is well known that susceptibility and resistance to disease present the most challenging phenotypes to study. This approach will not only answer fundamental questions of host-pathogen interactions, but will also aid in further improving the transcriptional annotation of the turkey genome. Also, the whole genome sequence serves as the anchor and coordinating platform for genome-wide SNP discovery and the future development of SNP-chip resources under development by colleagues at the USDA (Beltsville). The initial sequence identified 920,126 single nucleotide variants of which 601,490 were considered as having strong support. One ultimate goal of these projects is to provide the community with robust tools and resources to facilitate immediate and long term applications of genome-wide selection for specific traits including production, disease resistance, and reproduction parameters. The most current sequence covers about 95% of the turkey genome and should yield a much improved gene annotation level, making it a valuable resource for comparative genomics and to study genetic variations underlying economically important quantitative traits in poultry.
Publications
- Peer Reviewed: Parra ZE, Mitchell K, Dalloul RA, Miller RD. A second TCR-delta locus in Galliformes uses antibody-like V domains: insight into the evolution of TCR-delta and TCR-mu genes in tetrapods. The Journal of Immunology. Published online March 9, 2012, doi: 10.4049/jimmunol.1103521
- Reed KM, Dalloul RA. The turkey genome sequence: implications for enhancing turkey biology. In: Turkey production and Health: An Update. Proceedings of the 6th International Meeting of the Working Group 10 (Turkey) of WPSA (Ed. Hafez, H.M), Berlin. Mensch & Buch Verlag, Germany. ISBN 978-3-86387-094-2. Pp. 23-29. 2011.
- Published Abstracts and Presentations: Monson MS, Settlage RE, Rawal S, Coulombe RA, Reed KM. Spleen transcriptome response to aflatoxin in the domestic turkey. Plant and Animal Genome XX, San Diego, CA. January 13-18, 2012.
- Kim S, Smith EJ, Reed KM, Miska KB, Dalloul RA. Evolution of nucleotide-binding oligomerization domain protein 2 (Nod2). Plant and Animal Genome XX, San Diego, CA. January 13-18, 2012.
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Progress 12/15/09 to 12/14/10
Outputs
OUTPUTS: As the Turkey Genome Sequencing Consortium, we used a combination of two next-generation sequencing platforms, Roche 454 and Illumina GAII, and unique assembly tools to sequence the genome of the agriculturally important turkey, Meleagris gallopavo. We have completed and made available the first draft genome sequence of the domestic turkey. Public outlets include NCBI GenBank (http://www.ncbi.nlm.nih.gov/genomeprj/42129), EBI Ensembl (http://uswest.ensembl.org/Meleagris_gallopavo/Info/Index), and the newly developed turkey GBrowse (http://128.175.126.109/cgi-bin/gbrowse/turkey10). The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. PARTICIPANTS: Rami A. Dalloul, Principal Investigator/Project Director; Kent M. Reed, Ed J. Smith, and Otto Folkerts, co-investigators. TARGET AUDIENCES: Poultry researchers (academic and industry) and producers, turkey breeders, and avian scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
A synergistic combination of two next-generation sequencing platforms with a detailed comparative BAC physical contig map provided a cost-effective assembly of the genome sequence of the domestic turkey (Meleagris gallopavo). Heterozygosity of the sequenced source genome allowed discovery of more than 600,000 high quality single nucleotide variants. Despite this heterozygosity, the current genome assembly (1.1 Gb) includes 917 Mb of sequence assigned to specific turkey chromosomes. Annotation identified nearly 16,000 genes, with 15,093 recognized as protein coding and 611 as non-coding RNA genes. Comparative analysis of the turkey, chicken, and zebra finch genomes, and comparing avian to mammalian species, supports the characteristic stability of avian genomes and identifies genes unique to the avian lineage. Clear differences are seen in number and variety of genes of the avian immune system where expansions and novel genes are less frequent than examples of gene loss. The turkey genome sequence provides resources to further understand the evolution of vertebrate genomes and genetic variation underlying economically important quantitative traits in poultry. This integrated approach may be a model for providing both gene and chromosome level assemblies of other species with agricultural, ecological, and evolutionary interest.
Publications
- Peer Reviewed (2010): Dalloul R.A., Long J.A., Zimin A.V., Aslam L., Beal K., Blomberg L.A., Bouffard P., Burt D.W., Crasta O., Crooijmans R.P., Cooper K., Coulombe R.A., De S., Delany M.E., Dodgson J.B., Dong J.J., Evans C., Frederickson K.M., Flicek P., Florea L., Folkerts O., Groenen M.A., Harkins T.T., Herrero J., Hoffmann S., Megens H.J., Jiang A., de Jong P., Kaiser P., Kim H., Kim K.W., Kim S., Langenberger D., Lee M.K., Lee T., Mane S., Marcais G., Marz M., McElroy A.P., Modise T., Nefedov M., Notredame C., Paton I.R., Payne W.S., Pertea G., Prickett D., Puiu D., Qioa D., Raineri E., Ruffier M., Salzberg S.L., Schatz M.C., Scheuring C., Schmidt C.J., Schroeder S., Searle S.M., Smith E.J., Smith J., Sonstegard T.S., Stadler P.F., Tafer H., Tu Z.J., Van Tassell C.P., Vilella A.J., Williams K.P., Yorke J.A., Zhang L., Zhang H.B., Zhang X., Zhang Y., Reed K.M. 2010. Multi-platform Next Generation Sequencing of the domestic turkey (Meleagris gallopavo): genome assembly and analysis. PLoS Biology 8:e1000475. (http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pb io.1000475)
- Published Abstracts and Presentations (2010):
- Dalloul R.A. Sequencing, assembly, and preliminary analysis of the turkey genome draft sequence. Poultry Workshop, Plant and Animal Genome XVIII Conference, San Diego, CA. January 9-13, 2010.
- Dalloul R.A., Burt D.W., Reed K.M., Folkerts O., Kaiser P., Smith E.J., Coulombe R.A., Dodgson J.B. Analysis of the turkey genome sequence reveals new insights into avian immune systems. Annual meeting, American Association of Avian Pathologists, Atlanta, GA. July 31-August 4, 2010.
- Burt D.W., Dalloul R.A. and the Turkey Genome Sequencing Consortium. The turkey genome. The 32nd conference of the International Society of Animal Genetics, Edinburgh, U.K. July 26-30, 2010.
- Burt D.W., Dalloul R.A. and the Turkey Genome Sequencing Consortium. The turkey genome. The Avian Immunology Research Group (AIRG) meeting, Budapest, Hungary. October 7-10, 2010.
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