Pilot project: Resequencing 3K sheep targets for SNPs and genotyping with the 1.5K Illumina sheep SNP array
 Two strategies have been considered for creating a high density sheep SNP chip - Sanger resequencing and next generation sequencing methods. The Sanger resequencing and subsequent pilot is outlined below. The pilot project was designed to test the final methodology required to get best recovery of SNPs per unit sequence and cost, examining pooling and various types of amplicons.

Step 1. Identify targets for resequencing
 Two subsets of the sheep genome were available for resequencing:
a) 370 K BAC end sequences (ISGC - USDA, AWI and MLA)
b) 140 K expressed sequences tags (ESTs - Ovita)
Target selection was based on
  • targets being free of repetitive sequence
  • equal spacing across the genome
  • association with some known genes
Primers were designed using an automated pipeline.

Step 2. Sequence through a panel of animals

 1. Texel - BAC library DNA donor (USDA Tim Smith)
2. NZ Romney (AgResearch, John McEwan)
3. Poll Dorset (sheepGENOMICS, Hutton Oddy, ram provided by George Carter)
4. Merino (sheepGENOMICS, Hutton Oddy, ram provided by UNE)
5. Lacaune (INRA, Andre Eggen)
6. Gulf Coast Native (USU, Noelle Cockett & Jim Miller)
7. Katahdin (USU, Noelle Cockett & Jim Miller)
8. Red Maasai (ILRI, Olivier Hanotte)
9. Awassi (University of Sydney, Herman Raadsma)
10. a pool of the above 9 sheep
11 and 12. 2 pools of 24 sheep per pool (pool 1 Texel/Romney, pool 2 Merino/Poll Dorset)
The collection of DNA samples from the animals and pools was completed in June 2007. The resequencing component of the pilot phase for 3,000 amplicons was then performed by the AGRF, and was completed in February 2007.

Step 3. Identify SNPs and design and create a SNP chip

 The project resulted in 49,077 useable sequences (deposited in GenBank GSS and Trace archives) with around 1 SNP being detected for every ~250 bp of sequence.
About 50% of these SNPs had properties of interest in terms of appropriate spacing across the genome, a minor allele frequency greater than 0.15, and the ability to be multiplexed into a SNP chip
sequencing
Figure 1. SNPs identified from processing trace files
SNP chip
Figure 2. SNP chips can query many thousands of loci at one time
Step 4. 1.5K Illumina sheep SNP array and genotyping of selected sheep resources

 A 1.5K pilot SNP chip was then designed, for more details click on this link. The pilot chip was then tested on 651 sheep from a range of breeds and various resources (including the IMF and UTAH5000 sheep radiation hybrid panel) using Illumina technology at Johns Hopkins University and University of Alberta. The work was completed in late 2007 and its analysis is currently underway. Formal publication of results from this work is expected in 2008.

Summary

 The information gained from the pilot resequencing project demonstrated that the Sanger resequencing strategy was successful but identified that it was impractical (both timewise and expensewise) for large scale SNP discovery in sheep. Consequently, the ISGC has decided to use the cheaper Roche 454 and Illumina Solexa whole genome shotgun and reduced representational sequencing instead of Sanger resequencing. However, the Sanger resequencing project and subsequent genotyping provided valuable information on sequence and breed diversity which is being used to design subsequent projects such as the sheep HapMap project .

Fortnightly phone discussions (Tuesday 9:00 Australian Eastern Standard Time) are held that discuss the progress of this work. To participate in these discussions contact James Kijas.

Last modified: 4th January 2008
Maintainers: John McEwan, Agresearch, Jill Maddox, University of Melbourne
Email: jillm@rubens.its.unimelb.edu.au