1. SNP Genotyping Santiago C. González-Martínez Center of Forest Research, INIA, PO Box 8111 28080 Madrid, Spain santiago@inia.es

2. Syvänen (2001) Nature Genetics

3. DGGE and SSCP

5. (eco)Tilling

6. Single-base extension

7. Fluorescence Polarization with Terminator-Dye Incorporation (FP-TDI) - a method for SNP detection

8. Chen et al. 1999 Genome Res. Fluorescence polarization

9. FP-TDI Assay

10. What the data might look like… if you are lucky Gene 4CL

11. Usually, the data is not quite as ‘perfect’… Gene EREBPS

12. FP-TDI ADVANTAGES No special primers Relatively simple procedure Results are immediate DISADVANTAGES Expensive plate reader = $30, 000 kit = $10, 000 for 100, 000x plates = $6 each cost per reaction = 75c each thermocycler maintenance PCR (ie mostly Taq) costs One SNP per reaction Need to know sequence

13. Pyrosequencing

14. Microarrays Syvänen (2001) Nature Genetics

15. High-throughput methods Syvänen 2005 Nature Genetics

16. Illumina- BeadStation500G-BeadLab Platform

17. How does it work?

18. Experimental Procedure

20. Example of a “nice” SNP

21. Examples of midrange SNPs Above threshold: GT = 0.7849 Cluster_sep =0.272 Below threshold: GT = 0.7788 Cluster_sep =0.267

22. 4CL-s5[c/t]

23. AT-s3[g/a] Not resolved with FPBetter separation in Illumina

24. Illumina- BeadStation demo