1. SeattleSNPs Variation Discovery Resource Materials prepared by: Mary E. Mangan, PhD www.openhelix.com Updated: Q1 2011 Version 1

2. Copyright OpenHelix. No use or reproduction without express written consent2 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

3. Copyright OpenHelix. No use or reproduction without express written consent3 Introduction Human Genome Project: the “reference” sequence Variation among humans is informative Projects to identify variations have been launched From http://www.genome.gov/10001772 From http://www.ncbi.nlm.nih.gov/genome/guide/human/ GenBank: http://www.ncbi.nlm.nih.gov/Genbank/index.html MapViewer: http://www.ncbi.nlm.nih.gov/mapview Ensembl: http://www.ensembl.org UCSC Genome Browser: http://genome.ucsc.edu

4. Copyright OpenHelix. No use or reproduction without express written consent4 SNPs: Single Nucleotide Polymorphisms SNP: Single Nucleotide Polymorphism SNPs may be: A single nucleotide change (A/G, as shown above) A small insertion or deletion (indels) SNPs may have no impact, or may cause disease SNPs can tell us about inheritance patterns Human ApoE gene segment, rs11542035 SNP GTACCGCGGCGC GTACCACGGCGC Reference sequence: Variant found: HIS ARG

5. Copyright OpenHelix. No use or reproduction without express written consent5 NHLBI Program for Genomic Applications NHLBI has special sub-programs, like PGA PGA mission: resources, reagents, educate, disseminate http://www.nhlbi.nih.gov/resources/pga/index.htm

6. Copyright OpenHelix. No use or reproduction without express written consent6 SeattleSNPs SeattleSNPs mission: identify, genotype, model SNPs Focus: inflammatory responses in humans Provides data and workshops, available to all Genotyping services also available SNP Discovery Candidate Gene Reqsequencing and Analysis SNP Genotyping Collaborative Genotyping Large-scale Association Studies SeattleSNPs Education Workshops Scientific Presenations

7. Copyright OpenHelix. No use or reproduction without express written consent7 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

8. Copyright OpenHelix. No use or reproduction without express written consent8 SeattleSNPs Team Team is lead by Drs. Deborah Nickerson and Mark Rieder Many people contribute to providing the data, software and support; see publications http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrie ve&dopt=AbstractPlus&list_uids=14681826&query_hl=3&itool=pubmed _docsum

9. Copyright OpenHelix. No use or reproduction without express written consent9 Candidate Gene Selection Select gene of interest Obtain longest sequenceRe-sequence genomic samples Heart, lung, blood research pathways of interest Search for longest gene model sequence Resequencing is performed

10. Copyright OpenHelix. No use or reproduction without express written consent10 Genomic Samples Obtained Genomic samples Find polymorphisms Provide data visualization, analysis and downloads Protocols: http://pga.gs.washington.edu/protocols.html Obtain genomic samples (now using HapMap) Sequence samples, identify polymorphismsAssemble data for viewing and downloading

11. Copyright OpenHelix. No use or reproduction without express written consent11 Sequence each end of the fragment. Sequence Amplify DNA 5’3’ Customized software tools Primer design algorithm Custom LIMS and database to track all aspects of data production and quality Robotics used to automate sample handling Base-calling Quality determination Contig assembly Final quality determination Sequence viewing Polymorphism tagging Polymorphism reporting Individual genotyping Polymorphism detection PolyPhred Consed Analysis Phred Phrap Data publication to WWW Sequencing Production & Data Analysis Pipeline

12. Copyright OpenHelix. No use or reproduction without express written consent12 Re-sequencing Pipeline Gene design-automated primer picking software All approaches 2 kb upstream of first exon, 2 kb downstream of last exon Gene is < 25 kb - Full: complete re-sequencing Gene is > 25 kb i.e. exons, conserved non-coding sequences, and sampling across intron sequences Prior to amplification and re-sequencing, problematic GC-rich regions, alu repeats, polynucleotide tracts, and pseudogenes identified Sequence in base-pairs Mapping of PCR primers Mapping of Exons Mapping of PCRs

13. Copyright OpenHelix. No use or reproduction without express written consent13 Re-sequencing Pipeline Universal primer sequences standardize sequencing reaction conditions Standard dye terminator sequencing chemistry Optimized for reaction volume and dilution Automated sequencing capillary electrophoresis

14. Copyright OpenHelix. No use or reproduction without express written consent14 Data Analysis Sequence each end of the fragment. Sequence Amplify DNA 5’3’ Customized software tools Primer design algorithm Custom LIMS and database to track all aspects of data production and quality Robotics used to automate sample handling Base-calling Quality determination Contig assembly Final quality determination Sequence viewing Polymorphism tagging Polymorphism reporting Individual genotyping Polymorphism detection PolyPhred Consed Analysis Phred Phrap Data publication to WWW

15. Copyright OpenHelix. No use or reproduction without express written consent15 Polymorphism Identification and Analysis individuals in rows SNPSNP

16. Copyright OpenHelix. No use or reproduction without express written consent16 Homozygous C/C Heterozygous C/T Homozygous T/T Polymorphisms

17. Copyright OpenHelix. No use or reproduction without express written consent17 Program for Early Career Investigators Apply for free genotyping and analysis http://pga.gs.washington.edu/genotyping.html

18. Copyright OpenHelix. No use or reproduction without express written consent18 In This Tutorial We will examine how to find genes of interest We will explore and understand the data and displays

19. Copyright OpenHelix. No use or reproduction without express written consent19 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

20. Copyright OpenHelix. No use or reproduction without express written consent20 Finding Data from SeattleSNPs http://pga.gs.washington.edu 2 main strategies Whole site search Gene lists site search for anything Browsers: Firefox Safari Explorer

21. Copyright OpenHelix. No use or reproduction without express written consent21 Sequencing Resources for Data Access Access lists of genes Access summaries

22. Copyright OpenHelix. No use or reproduction without express written consent22 Finding Genes From the Sequenced Genes list Find genes and panel info

23. Copyright OpenHelix. No use or reproduction without express written consent23 Gene List Information Gene list options

24. Copyright OpenHelix. No use or reproduction without express written consent24 Coriell DNA Panels p1: Coriell CEPH/AA panel p2: Coriell HapMap European/African panel from HapMap http://www.coriell.org/ Yoruba in Ibadan Nigeria CEPH European Ancestry Utah Centre d’Etude du Polymorphisme Humain African American DNA

25. Copyright OpenHelix. No use or reproduction without express written consent25 Panels Integrate with Other Data p1 panels integrate with Perlegen data Hinds et al. Science 2005. Whole-genome patterns of common DNA variation in three human populations. p2 panels integrate with HapMap data The International HapMap Consortium. A haplotype map of the human genome. Nature 2005. p1 DNA panel - Perlegen Integration (1.58 million SNPs) = SeattleSNPs (1/200 bp)= Perlegen SNPs (~1/3000 bp) p2 DNA panel - HapMap Integration (~3.5 million SNPs) = SeattleSNPs (1/200 bp)= HapMap SNPs (~1/1000 bp)

26. Copyright OpenHelix. No use or reproduction without express written consent26 Panels Integrate with Other Data SeattleSNPs provides much more density of SNPs p1 panels integrate with Perlegen data p2 panels integrate with HapMap data SeattleSNPs DataHapMap

27. Copyright OpenHelix. No use or reproduction without express written consent27 Gene List Information Gene list options

28. Copyright OpenHelix. No use or reproduction without express written consent28 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

29. Copyright OpenHelix. No use or reproduction without express written consent29 SeattleSNPs Displays Gene-specific page Image of gene structure and SNPs Links to other resources and download Access gene-specific details and data gene, location

30. Copyright OpenHelix. No use or reproduction without express written consent30 Understanding SeattleSNPs Images Gene structure: exons, introns, UTRs coordinates based on their GenBank submission of this gene SNPs Controls for changing the view SeattleSNPs coordinates Gene structureSNPs change view SNP choices

31. Copyright OpenHelix. No use or reproduction without express written consent31 Gene-Specific Links Links to other resources UCSC Custom track shows the SeattleSNPs  custom tracks 

32. Copyright OpenHelix. No use or reproduction without express written consent32 SeattleSNPs Data Types Alox12: download all data Populations genotyped for this gene

33. Copyright OpenHelix. No use or reproduction without express written consent33 SeattleSNPs Data Types Documentation for all data types on the left Links to all the data on the right DocumentationData

34. Copyright OpenHelix. No use or reproduction without express written consent34 Mapping Data Mapping data types GenBank record for SeattleSNP coordinate system

35. Copyright OpenHelix. No use or reproduction without express written consent35 Mapping Data Mapping data types GenBank record for SeattleSNP coordinate system

36. Copyright OpenHelix. No use or reproduction without express written consent36 Genotyping Data Genotyping data for individuals individuals site of variation, 5’  3’

37. Copyright OpenHelix. No use or reproduction without express written consent37 Genotyping Data Genotyping data for individuals

38. Copyright OpenHelix. No use or reproduction without express written consent38 Linkage Data Linkage data “Tag” SNPs that can be used for genotyping See Carlson et al., Am. J. Hum. Genet., 74:106-120, 2004

39. Copyright OpenHelix. No use or reproduction without express written consent39 LDSelect: Using LD to Pick tagSNPs LDSelect Uses SNP discovery data (not haplotypes) Finds all correlated SNPs to minimize the total number Maintains genetic diversity of locus Carlson et al. AJHG (2004)

40. Copyright OpenHelix. No use or reproduction without express written consent40 “…a unique combination of genetic markers present in a chromosome.” pg 57 in Hartl & Clark, 1997 Multi-SNP Correlations (aka Haplotypes)

41. Copyright OpenHelix. No use or reproduction without express written consent41 Haplotyping Data PHASE algorithm: infer haplotype statistically Stephens, et al. Am J Hum Genet. 2001 PHASE: http://www.stat.washington.edu/ stephens/software.html

42. Copyright OpenHelix. No use or reproduction without express written consent42 Haplotyping Data Haplotyping data Visual Haplotype: upload your own data OR select gene from list upload data

43. Copyright OpenHelix. No use or reproduction without express written consent43 Haplotyping Data Visual Haplotype data output Alox12 individuals site of variation

44. Copyright OpenHelix. No use or reproduction without express written consent44 Predictive Analysis Predictive analysis on non-synonymous SNPs SIFT: Ng and Henikoff, Gen. Research, 12:436-446, 2002 PolyPhen: Ramensky, et al., NAR 30:17:3894-3900, 2002 Sorting Intolerant From Tolerant (http://blocks.fhcrc.org/sift/SIFT.html) PolyPhen (http://coot.embl.de/PolyPhen)

45. Copyright OpenHelix. No use or reproduction without express written consent45 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

46. Copyright OpenHelix. No use or reproduction without express written consent46 Downloading SeattleSNPs Data Download all the data Can also download just 1 gene from the gene page Usage/Citation policy: http://pga.gs.washington.edu/UseStatement.html all data subsets gene page

47. Copyright OpenHelix. No use or reproduction without express written consent47 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

48. Copyright OpenHelix. No use or reproduction without express written consent48 Workshop Information Workshops offered in Seattle Slides, materials available

49. Copyright OpenHelix. No use or reproduction without express written consent49 Traveling Workshops Bring SeattleSNPs to your site

50. Copyright OpenHelix. No use or reproduction without express written consent50 Recorded Tutorial and Quick Reference Cards Recorded tutorial Download materials Order Quick Reference Cards

51. Copyright OpenHelix. No use or reproduction without express written consent51 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

52. Copyright OpenHelix. No use or reproduction without express written consent52 Summary SeattleSNPs PGA program; focus on heart, lung, blood genes Genotypes, haplotypes, web access and downloads Educational resources

53. Copyright OpenHelix. No use or reproduction without express written consent53 SNP Data SeattleSNPs data available in several ways Other projects exist to identify SNP variations Project scope, population, and methods may vary dbSNP database +

54. Copyright OpenHelix. No use or reproduction without express written consent54 SeattleSNPs Agenda Introduction SeattleSNPs Process Basic Searches Understanding the Displays Downloads Education Summary Exercises http://pga.gs.washington.edu

55. Copyright OpenHelix. No use or reproduction without express written consent55