Progress 04/01/12 to 09/30/13
Outputs
Target Audience: University faculty and students Fellow participants in Regional Project NC-1170 Genearl poultry scientific community Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This project involved particpation by a graduate student (Melissa Monson) for data analysis, and an two undergraduate students (Molly Greenshields and Adam Busaker) who performed lab experiments. How have the results been disseminated to communities of interest? Oral and poster presentations at regional and international conferences and through University seminar presentations. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Aim 1.Whole genome sequencing of the Ocellated turkey. To assemble the genome we have obtained sequence reads from two platforms. First, high molecular weight DNA was submitted to the BMGC at the Univeristy of Minnesota for Illumina sequencing. The sequencing library was prepped using the longer length protocol (~500bp). Approximately 550M paired-end reads (2 x 100bp) were generated from a random sheared library on the Illumina HighSeq 2000 (3 flow cell lanes). This represents almost 60x genome coverage. In addition, ~1.6M paired-end reads were generated from a large insert library (3-5 kb) on the Roche 454 platform at Virginia Tech University. A second large insert library (~8kb) was also sequenced. Sequence assembly is being conducted at the Virginia Bioinformatics Institute (with collaborator Dr. Robert Settlage) by alignment of reads with the latest build of the domestic turkey genome (UMD5.0). In addition, the domestic turkey MHC sequence was used to identify sequence reads within the Illumina data set to assemble the MHC of the ocellated turkey. The majority of this sequence have now been verified and oriented using PCR and direct sequencing. We are still completing data analysis and manuscript preparation. Aim 2. Development of an MHC sequence capture array and comparative MHC sequencing. Working with the Agilent SureSelect design team, a platform of over 12,000, 120mer cRNA baits was designed to capture MHC sequence for next-generation sequencing. Sheared genomic DNA libraries were created for 16 individuals including 14 turkeys (domestic, heritage and wild), an ocellted turkey (a second bird other than used above), and a chicken, and hybridized to the cRNA baits. Recovered fragments were subjected to automated sequencing (151bp paired-end run on the Illumina MiSeq platform). Included in our samples was the DNA the bird used to sequence the turkey genome ("Nici") and the chicken genome (RJF-256). The remaining birds were chosen from those previously determined to have diverse MHC haplotypes. M. ocellata and chicken (Gallus gallus) samples were included to determine the applicability of the custom platform to related species. Sequencing of the library pool has been completed (~6M reads representing 309K-458K reads/library). Initial analysis of this data indicates good sequence recovery with average match to the turkey genome exceeding 85% and match to MHC in excess of 50%. From this data we have a assembled a large array of SNPs for all individuals examined. The results of this aim are being summarized for publication.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Monson M, Mendoza KM, Settlage R, and Reed KM. Initial assembly and analysis of the MHC B-locus of the ocellated turkey. Plant, Animal and Microbial Genomes Conference XXI, San Diego, CA. Poster
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Reed KM, Monson M, Settlage RE, Mendoza KM and RA Dalloul. MHC composition and variation in turkeys of the Americas. 100th annual meeting of the American Association of Immunologists, Honolulu, HI. Poster
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2013
Citation:
Reed KM. Use of a capture platform for target enrichment and massively parallel sequencing to assess variation in the turkey MHC. Oral presentation NC1170 Poultry Workshop Plant & Animal Genome XXI
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Our plan is to create a high density whole genome sequence of the Ocellated turkey (Aim 1) in conjunction with MHC-specific sequencing (Aim 2) using the newly developed SureSelect target enrichment system (Agilent). Because of the constraints placed on release of the first year of funding, the time line for the projects was adjusted to focus collection of sequence data for both aims in year 1 leaving year 2 for data analysis. Aim 1. Whole genome sequencing of the Ocellated turkey. To assemble the genome we have obtained sequence reads from two platforms. First, high molecular weight DNA was submitted to the BMGC at the Univeristy of Minnesota for Illumina sequencing. The sequencing library was prepped using the longer length protocol (~500bp). Approximately 550M paired-end reads (2 x 100bp) were generated from a random sheared library on the Illumina HighSeq 2000 (3 flow cell lanes). This represents almost 60x genome coverage. In addition, ~1.6M paired-end reads were generated from a large insert library (3-5 kb) on the Roche 454 platform at Virginia Tech University. A second large insert library (~8kb) is currently being sequenced and should be completed in the next 2 weeks. Sequence assembly will be conducted independently at the Virginia Bioinformatics Institute (with collaborator Dr. Robert Settlage) and at the University of Maryland (collaborator Dr. Aleksy Zimin). We have already used the domestic turkey MHC sequence to identify sequence reads within the Illumina data set and are working to assemble the MHC of the ocellated turkey. This sequence will be verified and oriented using PCR and direct sequencing. Aim 2. Development of an MHC sequence capture array and comparative MHC sequencing Working with the Agilent SureSelect design team, a platform of over 12,000, 120mer cRNA baits was designed to capture MHC sequence for next-generation sequencing. Sheared genomic DNA libraries were created for 16 individuals including 14 turkeys (domestic, heritage and wild), an ocellted turkey, and a chicken, and hybridized to the cRNA baits. Recovered fragments were subjected to automated sequencing (151bp paired-end run on the Illumina MiSeq platform). Included in our samples was the DNA the bird used to sequence the turkey genome ("Nici") and the chicken genome (RJF-256). The remaining birds were chosen from those previously determined to have diverse MHC haplotypes. M. ocellata and chicken (Gallus gallus) samples were included to determine the applicability of the custom platform to related species. Sequencing of the library pool has been completed (~6M reads representing 309K-458K reads/library). Initial analysis of this data indicates good sequence recovery with average match to the turkey genome exceeding 85% and match to MHC in excess of 50%. PARTICIPANTS: Kent M. Reed, Department of Veterinary and Biomedical Sciences, University of Minnesota. Role: project PD. Kristelle M. Mendoza, Department of Veterinary and Biomedical Sciences, University of Minnesota. Role: laboratory scientist. Melissa Monson, Department of Veterinary and Biomedical Sciences, University of Minnesota. Role: graduate student, data analysis. Molly Greenshields, Department of Veterinary and Biomedical Sciences, University of Minnesota. Role: Undergraduate laboratory assistant. Robert Settlage, Virginia Bioinformatic Institute, Virginia Tech University Role: collaborator, data analysis. Aleksey Zimin, University of Maryland Role: collaborator, genome analysis. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Because of the constraints placed on the first year of funding, the time line for the project was adjusted to focus collection of sequence data for both aims 1 and 2 in year 1 leaving year 2 for data analysis.
Impacts
The results of this project will generate the genome sequence of a second turkey species (Ocellated) and a detailed assembly of the Major Histocompatibility Complex (MHC) region (Aim 1). Re-sequencing of multiple individuals through focused sequence capture (Aim 2) will provide a means to test for copy number variation in the MHC for the turkey. Locus remodeling (gene rearrangement) and copy number variation (CNV) within the MHC are increasingly identified as having significant impact on disease susceptibility/resistance.
Publications
- No publications reported this period
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