Resources at HapMap.Org HapMap3 Tutorial Marcela K. Tello-Ruiz Cold Spring Harbor Laboratory

Low LD -> Recombination Basic Concepts Parent 1 Parent 2 A B a b A B a b X A B a b A b A B A B a b OR a B A B A b a b A B a B A b A B a b A B etc… High LD -> No Recombination (r2 = 1) SNP1 “tags” SNP2 Low LD -> Recombination Many possibilities

Basic Concepts SNP1 SNP2 alleles: A/a B/b C1 C2 POP allele freqs: A (80%) B (60%) a (20%) b (40%) genotypes: Person 1 Person 2 Person 3 AA AA Aa BB Bb Bb phased haplotypes (C1/C2): A B A B A B A B A b a b OR A b a B

HapMap Glossary LD (linkage disequilibrium): For a pair of SNP alleles, it’s a measure of deviation from random association (i.e., no recombination). Measured by D’, r2, LOD Phased haplotypes: Estimated distribution of SNP alleles. Alleles transmitted from Mom are in same chromosome haplotype, while Dad’s form the paternal haplotype. Tag SNPs: Minimum SNP set to identify a haplotype. r2= 1 indicates two SNPs are redundant, so each one perfectly “tags” the other. Questions? help@hapmap.org

HapMap International Consortium HapMap Project Phase 1 Phase 2 Phase 3 Samples & POP panels 269 samples (4 panels) 270 samples 1,115 samples (11 panels) Genotyping centers HapMap International Consortium Perlegen Broad & Sanger Unique QC+ SNPs 1.1 M 3.8 M (phase I+II) 1.6 M (Affy 6.0 & Illumina 1M) Reference Nature (2005) 437:p1299 Nature (2007) 449:p851 Draft Rel. 1 (May 2008)

3.9 M unique QC+ SNPs -- > 1 SNP/700 bp Release Notes Phase 1+2: Latest Release #24, October 2008 (NCBI build 36): 3.9 M unique QC+ SNPs -- > 1 SNP/700 bp http://ftp.hapmap.org/00README.releasenotes_rel24 Added back chrX SNPs dropped in previous releases Corrected allele flips from rel#23a Phase 3: Draft release #1 (NCBI build 36) http://ftp.hapmap.org/genotypes/2008-07_phaseIII/00README.txt HapMap3 sites @ Broad Institute, Sanger Center and Baylor College

Phase 3 Samples * Population is made of family trios

Phase 3 11 panels & 1,115 samples Platforms: 558/557 males/females 924/191 founders/non-founders Platforms: Illumina Human 1M (Sanger) Affymetrix SNP 6.0 (Broad) EXCLUDED from QC+ data set: Samples with low completeness, and SNPs with low call rate in each pop (< 80%) and not in HWE (p < 0.001) Overall false positive rate: ~3.2% Data merged with PLINK (concordance over 249,889 overlapping SNPs = 0.9931) Alleles on the (+/fwd) strand of NCBI b36

Phase 3: Draft Release 1 samples QC+ SNPs poly QC+ SNPs 71 ASW 1,632,186 1,536,247 162 CEU 1,634,020 1,403,896 82 CHB 1,637,672 1,311,113 70 CHD 1,619,203 1,270,600 83 GIH 1,631,060 1,391,578 82 JPT 1,637,610 1,272,736 83 LWK 1,631,688 1,507,520 71 MEX 1,614,892 1,430,334 171 MKK 1,621,427 1,525,239 77 TSI 1,629,957 1,393,925 163 YRI 1,634,666 1,484,416

Phase 3 Data HapMap format: http://ftp.hapmap.org/genotypes/2008-07_phaseIII/hapmap_format * Excluded 1,527 SNPs with strandedness issues & 411 indels PLINK format: http://ftp.hapmap.org/genotypes/2008-07_phaseIII/plink_format HapMap3 sites: Broad - http://www.broad.mit.edu/~debakker/p3.html Sanger - http://www.sanger.ac.uk/humgen/hapmap3/ Baylor - http://www.hgsc.bcm.tmc.edu/projects/human/

Goals of This Tutorial This tutorial will show you how to: Find HapMap3 SNPs near a gene or region of interest (ROI) Visualize allele frequencies in HapMap3 populations Download SNP genotypes in ROI for use in Haploview 4.1 Identify GWA hits in the vicinity of ROI & visualize in the context of all chromosomes (karyogram) Add custom data onto the GWAs karyogram Add custom tracks of association data onto ROI Create publication-quality images Download the entire HapMap3 data set in bulk Distinguish genotype data in PLINK and HapMap formats Visualize LD patterns, find tag SNPs, impute genotypes using release #24 (phase 1+2) Generate customized extracts of the entire dataset using HapMart

1: Surf to the HapMap Browser 1a. Go to www.hapmap.org 1b. Select “HapMap phase 3”

2. Type search term – “TCF7L2” 2: Search for TCF7L2 Search for a gene name, a chromosome band, or a phrase like “insulin receptor” 2. Type search term – “TCF7L2”

3: Examine Region Chromosome-wide summary data is shown in overview Region view puts your ROI in genomic context Default tracks show HapMap genotyped SNPs, refGenes with exon/intron splicing patterns, etc. 3: This exonic region has many typed SNPs. Click on ruler to re-center image.

3: Examine Region (cont) Use the Scroll/Zoom buttons and menu to change position & magnification 3: Mouse over a SNP to see allele frequency table As you zoom in further, the display changes to include more detail Click to go to SNP details page

4: Generate Text Reports 4: Select the desired “Download” option and press “Go” or “Configure” Available phase 3 downloads: - Individual genotypes - Population allele & genotype frequencies

4: Generate Reports (cont) The Genotype download format can be saved to disk or loaded directly into Haploview v4.1

5b: Find GWA hits in nearby region. Click on a GWA hit to re-center 5a: Scroll down to turn on GWA studies tracks in overview & region panels 5b: Find GWA hits in nearby region. Click on a GWA hit to re-center

5c: Mouse over & click on GWA hit for more info 5: Find GWA hits (cont) 5c: Mouse over & click on GWA hit for more info

6: Examine GWA hits in entire genome 6: From www.hapmap.org, select “Karyogram”

6: Custom GWA hits in karyogram 6: Follow these instructions to upload your own GWA data Detailed help on the format is under the “Help” link

7: Create your own tracks Example: Interested in T2DM genetics Create file with custom annotations from http://www.broad.mit.edu/diabetes and superimpose on the HapMap 7: Upload example file: TCF7L2_annotations.txt Detailed help on the format is under the “Help” link

7: Create your own tracks (cont) Some SNPs were typed (known platform) and others were imputed. Format data for both typed & imputed SNPs. Scores allow you to display data in quantitative form, such as XY plots Save as a text file!

7: Create your own tracks (cont) Remember to point your browser to the location of your annotations (TCF7L2 gene in this case).

7: Create your own tracks (cont) Make edits on your own browser window by clicking on “Edit File…”

7: Create your own tracks (cont)

8: Create Image for Publication Click on the +/- sign to hide/show a section 8a. Click on “High-res Image” Mouse over a track until a cross appears. Click on track name to drag track up or down.

8: Image for Publication (cont) 8b. Click on “View SVG Image in new browser window” 8c. Save generated file with “.svg” extensions Can view file in Firefox, but use other programs (Adobe Illustrator or Inkscape) to convert to other formats and/or edit

8: Image for Publication (cont) Inkscape is free and lets you edit and convert to other formats (many journals prefer EPS)

9. Bulk downloads Or directly click on “Data” 18. From www.hapmap.org, click on “Bulk Data Download”

HapMap3 genotypes & frequencies 9. Bulk downloads Download the entire HapMap3 data set to your own computer HapMap3 genotypes & frequencies 9a. Select “Genotypes” Analytic results (LD & phased haplotype data available for HapMap3) Your own copy of the HapMap Browser Protocols & assay design HapMap Samples Also available at http://ftp.hapmap.org

9b. Click on hapmap_format/forward to download genotypes 9. Bulk downloads (cont) 9b. Click on hapmap_format/forward to download genotypes Also at http://ftp.hapmap.org/genotypes/latest_phaseIII_ncbi_b36/

10: Surf to the HapMap phase 1+2 genome browser 10. Go to www.hapmap.org & select “HapMap Genome Browser B36”

11. Type search term – “TCF7L2” 11: Search for TCF7L2 11. Type search term – “TCF7L2”

12: Examine Region 12. Re-center & zoom in

12: Turn on LD & Haplotype Tracks 12a: Scroll down to the “Tracks” section. Turn on the LD Plot and Haplotype Display tracks. 12b: Press “Update Image” These sections allow you to adjust the display and to superimpose your own data on the HapMap

13: View variation patterns Triangle plot shows LD values using r2 or D’/LOD scores in one or more HapMap populations Phased haplotype track shows all 120 chromosomes with alleles colored yellow and blue

14: Adjust Track Settings (on the spot) 14a. Click on question mark preceding track name 14b. Adjust population and display settings & press “Configure”

14: Adjust Track Settings (cont) Select the analysis track to adjust and press “Configure”

15: Activate the “tag SNP Picker” and press “Update Image” 15: Turn on Tag SNP Track 15: Activate the “tag SNP Picker” and press “Update Image”

16: Adjust tag SNP picker Tag SNPs are selected on the fly as you navigate around the genome Alternatively, you may select “Annotate tag SNP Picker” and press “Configure…” 16a: Click on question mark behind “tag SNP Picker”

16: Adjust tag SNP picker (cont) Select population Select tagging algorithm and parameters [optional] upload list of SNPs to be included, excluded, or design scores 16b: Press “Configure” to save changes

17: Impute genotypes using HapMap Data Interested in the VAV1 gene Commercially available platforms with few overlapping SNPs in this region HapMap genotyped lots of SNPs in region Use genotypes for HapMap SNPs to impute genotypes & compare non-overlapping SNP sets!

17: Impute genotypes using MACH1 17b. Select “Download Impute Data”, click “Configure” 17a. Go to chr19:6,765,000..6,900,000

17: Configure MACH1 17c. Upload input files: example.dat & example.ped. Enter e-mail address. Click “Go”

17: Impute genotypes: Input files example.dat (20 user-provided SNPs; all should be part of the HapMap): M rs4807101 M rs164022 M rs625828 M rs461970 M rs331684 … example.ped (genotypes for 336 unrelated inds): PED00001 IND00001 0 0 2 C/C C/C T/T C/T C/C G/G G/G … PED00002 IND00002 0 0 1 C/T C/C T/T T/T C/C A/A A/G … PED00003 IND00003 0 0 2 T/T G/G A/A C/T C/C A/G A/G …

17. Visualize imputed SNPs 17d. Return to browser 17. Visualize imputed SNPs Your imputation results appear as an external track that can be edited. Hint: Click on “Help” link below for display options 17e. Click “Edit File”

17. Edit external annotations file 17f. Edit annotations file & “Submit Changes”

17. Edit external annotations file

17: Impute genotypes: Results 17g. Check your e-mail for text results Info (143 provided & imputed HapMap SNPs) SNP Al1 Al2 Freq1 MAF Quality Rsq rs10419572 T A 0.9041 0.0959 0.8179 0.1069 rs415218 T A 0.9709 0.0291 0.9427 0.0313 rs4807100 A G 0.4713 0.4713 0.9790 0.9625 rs4807101 T C 0.4714 0.4714 0.9803 0.9649 rs1651876 T C 0.9631 0.0369 0.9277 0.0216 … Geno (143 SNPs x 336 inds) PED00001->IND00001 ML_GENO T/T T/T G/G C/C T/T T/T A/T G/G A/A T/T T/C … PED00002->IND00002 ML_GENO T/T T/T A/G T/C T/T T/T A/T G/G A/A T/T T/C … PED00003->IND00003 ML_GENO T/T T/T A/A T/T T/T T/T A/T G/G A/A T/T T/T … Dose (allele dosage) PED00001->IND00001 ML_DOSE 1.719 1.911 0.004 0.003 1.913 1.980 1.246 1.884 1.949 1.948 1.302 … PED00002->IND00002 ML_DOSE 1.861 1.957 1.000 1.000 1.952 1.892 1.086 1.909 1.949 1.948 1.096 … PED00003->IND00003 ML_DOSE 1.994 1.999 1.993 1.995 1.955 1.656 1.297 1.863 1.987 1.988 1.374… Probability of match imputed:experimental genotype (1.0 for provided markers)

18. Use HapMart to Generate Extracts of the HapMap Dataset Find all HapMap characterized SNPs that: Have a MAF > 0.20 in the Yoruban population panel (YRI) Cause a nonsynonymous amino acid change Were typed by Perlegen

Further Information HapMap Publications & Guidelines http://hapmap.cshl.org/publications.html.en Past tutorials & user’s guide to HapMap.org http://www.hapmap.org/tutorials.html.en Questions? help@hapmap.org

HapMap DCC Present Members (CSHL) Lincoln Stein Marcela K. Tello-Ruiz Zhenyuan Lu Wei Zhao HapMap DCC Former Members Lalitha Krishnan Albert Vernon Smith Gudmundur Thorisson Fiona Cunningham