Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1) Produce a draft reference genome sequence for rainbow trout. 2) Produce a high density SNP chip for rainbow trout. Approach (from AD-416): 1.a. Sequence the BACs from the physical map minimal tiling path (MTP) using the new Illumina sequencing platform. 1.b. Produce a dense genetic map using 5,000-10,000 SNPs from the Swanson x Whale Rock doubled haploid (DH) recombinant line of Gary Thorgaard to aid in the integration of the genetic and physical maps and the genome sequence assembly. 2.a. Add more SNPs to the current NCCCWA database of 25,000-50,000 putative SNPs using reduced representation sequencing approaches on the Thorgaard�s androgenetic DH lines and additional outbred populations of economic and scientific interest, and based on the genome sequence assembly from objective 1, select and design SNP markers for a chip of up to 50K SNPs. 2.b. Validate a subset of the SNPs using a smaller genotyping assay (e.g. Illumina�s 3K GoldenGate). 2.c. Produce a commercial high-density SNP assay for whole genome simultaneous genotyping in rainbow trout. Single Nucleotide Polymorphism (SNP) Discovery: A large dataset of high quality restriction associated DNA (RAD) SNPs that passed our rigorous analysis pipeline was generated from a panel of 11 homozygous doubled haploid rainbow trout lines as a resource for designing a high density SNP chip genotyping assay. The current bioinformatics pipeline for analyzing RAD sequence data was found to be very useful for assessing genetic diversity within a single rainbow trout population or between a pair of populations, but not for comparing more than two populations in a single analysis. The generally low percentage of RAD SNPs shared between populations in a pairwise comparison suggests that for a SNP chip to be useful for a wide range of genetic analyses in rainbow trout it will be important to select genetic markers having polymorphisms shared by as many populations as possible. SNP Chip Design: An Affymetrix Axiom chip design composed of 57,500 SNPs was completed using primarily the RAD SNPs dataset we generated from the homozygous rainbow trout lines but also by using other SNP datasets that were generated by US and international collaborators. Production, validation and evaluation of the 57K SNP chip is currently ongoing.
Impacts
(N/A)
Publications
|
Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): 1) Produce a draft reference genome sequence for rainbow trout. 2) Produce a high density SNP chip for rainbow trout. Approach (from AD-416): 1.a. Sequence the BACs from the physical map minimal tiling path (MTP) using the new Illumina sequencing platform. 1.b. Produce a dense genetic map using 5,000-10,000 SNPs from the Swanson x Whale Rock doubled haploid (DH) recombinant line of Gary Thorgaard to aid in the integration of the genetic and physical maps and the genome sequence assembly. 2.a. Add more SNPs to the current NCCCWA database of 25,000-50,000 putative SNPs using reduced representation sequencing approaches on the Thorgaard�s androgenetic DH lines and additional outbred populations of economic and scientific interest, and based on the genome sequence assembly from objective 1, select and design SNP markers for a chip of up to 50K SNPs. 2.b. Validate a subset of the SNPs using a smaller genotyping assay (e.g. Illumina�s 3K GoldenGate). 2.c. Produce a commercial high-density SNP assay for whole genome simultaneous genotyping in rainbow trout. This project has the overall goals of developing state of the art resources for genetic analyses in rainbow trout, primarily a genetic marker array that is constructed using information from research and commercial populations that are overlaid on the genome sequence, which must first be obtained. Significant progress was made in obtaining the genome sequence (Objective 1) and developing the genetic marker of choice, single nucleotide polymorphisms (SNP, Objective 2). Objective 1: Our strategy for sequencing the trout genome includes construction of a physical map built from small overlapping fragments of the genome which can easily be sequenced. This year we improved the physical map by adding over 20,000 new fragments to our analysis, providing a 13% increase in map information. The new map covers approximately 80% of the rainbow trout genome, of which we have sequence information for 95%; therefore the current draft of our sequence represents ~70% of the genome. A genetic map revealing the order of 5000 SNPs on trout chromosomes was constructed to aid in ordering the overlapping fragments of the physical map. Objective 2: Progress was made in identifying a set of over 109,000 SNPs from 8 research populations. The usefulness of this data set was validated on 18 populations from commercial and research sources to demonstrate the need for stringent marker selection criteria prior to construction of high-density genotyping platforms that aim to benefit all populations. To date this data set is the resource on which an international trout genotyping collaboration is being developed.
Impacts
(N/A)
Publications
|
Progress 10/01/10 to 09/30/11
Outputs
Progress Report Objectives (from AD-416) 1) Produce a draft reference genome sequence for rainbow trout. 2) Produce a high density SNP chip for rainbow trout. Approach (from AD-416) 1.a. Sequence the BACs from the physical map minimal tiling path (MTP) using the new Illumina sequencing platform. 1.b. Produce a dense genetic map using 5,000-10,000 SNPs from the Swanson x Whale Rock doubled haploid (DH) recombinant line of Gary Thorgaard to aid in the integration of the genetic and physical maps and the genome sequence assembly. 2.a. Add more SNPs to the current NCCCWA database of 25,000-50,000 putative SNPs using reduced representation sequencing approaches on the Thorgaard�s androgenetic DH lines and additional outbred populations of economic and scientific interest, and based on the genome sequence assembly from objective 1, select and design SNP markers for a chip of up to 50K SNPs. 2.b. Validate a subset of the SNPs using a smaller genotyping assay (e.g. Illumina�s 3K GoldenGate). 2.c. Produce a commercial high-density SNP assay for whole genome simultaneous genotyping in rainbow trout. A minimal tilling path (MTP) of approximately 15,000 BAC clones was extracted from the rainbow trout physical map and the MTP clones were picked from the original BAC libraries and re-arrayed into 96-well plates. DNA was extracted from each of the MTP clones and pooled in an indexed approach for tagging and sequencing with the Illumina sequencing platform. Three mate-pairs libraries were prepared from Swanson genomic DNA and sequenced to aid in assembling sequence scaffolds for the BACs using sequence assembly software programs. The assembly of sequence scaffolds is currently underway to enable producing a reference genome sequence assembly that will be guided by the rainbow trout physical map. In addition, a high density genetic map composed of approximately 5,000 SNPs and derived from the Swanson doubled haploid line was produced to aid in the ordering and assembly of the sequence scaffolds into chromosomes. We are also conducting a comprehensive SNP markers discovery and validation effort to characterize their polymorphism between and within rainbow trout populations using next-generation sequencing of Restriction-site Associated DNA (RAD) tags. Tissue samples were collected from 24 rainbow trout populations that are of economic and scientific interest and DNA is currently being extracted from those samples.
Impacts
(N/A)
Publications
|
|