1 of 25

Sequence Variation in EnsemblSequence Variation in Ensembl

2 of 25

OutlineOutline

• SNPs• SNPs in Ensembl• Linkage disequilibrium• SNPs in BioMart• DAS sources

3 of 25

Single nucleotide polymorphisms Single nucleotide polymorphisms (SNPs)(SNPs)

• Two human genomes differ by ~0.1%

• Polymorphism: a DNA variation in which each possible sequence is present in at least 1% of people

• Most polymorphisms (~90%) take the forms of SNPs: variations that involve just one nucleotide• ~1 out of every 300 bases in the human

genome• ~10 million in the human genome

4 of 25

Functional ConsequencesFunctional Consequences

• SNPs in coding area that alter aa sequence

• SNPs in coding areas that don’t alter aa sequence

• SNPs in promoter or regulatory regions

• SNPs in other regions

Cause of most monogenic disorders, e.g: Hemochromatosis (HFE) Cystic fibrosis (CFTR) Hemophilia (F8)

May affect splicing

May affect the level, location or timing of gene expression

No direct known impact on phenotype, useful as markers

5 of 25

Practical ApplicationsPractical Applications

• Disease diagnosis• Association studies• Pharmacogenomics• Forensic testing• Population genetics and

evolutionary studies• Marker-assisted selection

6 of 25

Practical ApplicationsPractical Applications

7 of 25

SNPs in EnsemblSNPs in Ensembl

• Most SNPs imported from dbSNP (rs……):• Imported data: alleles, flanking sequences, frequencies,

….• Calculated data: position, synonymous status, peptide

shift, ….

• For human also:• HGVbase• TSC• Affy GeneChip 100K and 500K Mapping Array• Affy Genome-Wide SNP array 6.0• Ensembl-called SNPs (from Celera reads and Jim

Watson’s and Craig Venter’s genomes)

• For mouse, rat, dog and chicken also:• Sanger- and Ensembl-called SNPs (other strains / breeds)

8 of 25

dbSNPdbSNP

• Central repository for simple genetic polymorphisms:• single-base nucleotide substitutions• small-scale multi-base deletions or insertions• retroposable element insertions and microsatellite

repeat variations

• http://www.ncbi.nlm.nih.gov/SNP/index.html

• For human (dbSNP build 128):• 34,434,159 submissions (ss#’s)• 11,883,685 RefSNP clusters (rs#’s)• 6,262,709 validated• 737,679 with frequency

9 of 25

SNPs in Ensembl - TypesSNPs in Ensembl - Types

Non-synonymous In coding sequence, resulting in an aa changeSynonymous In coding sequence, not resulting in an aa changeFrameshift In coding sequence, resulting in a frameshiftStop lost In coding sequence, resulting in the loss of a stop

codonStop gained In coding sequence, resulting in the gain of a stop codon

Essential splice site In the first 2 or the last 2 basepairs of an intronSplice site 1-3 bps into an exon or 3-8 bps into an intron

Upstream Within 5 kb upstream of the 5'-end of a transcriptRegulatory region In regulatory region annotated by Ensembl5' UTR In 5' UTRIntronic In intron3' UTR In 3' UTRDownstream Within 5 kb downstream of the 3'-end of a transcriptIntergenic More than 5 kb away from a transcript

10 of 25

SNPs in Ensembl - SpeciesSNPs in Ensembl - Species

• Human

• Chimp

• Mouse

• Rat

• Dog

• Cow

• Platypus

• Chicken

• Zebrafish

• Tetraodon

• Mosquito

11 of 25

CaveatCaveat

For human, mouse and rat Ensembl defines all SNP alleles respective to the + strand of the genome assembly! (to be able to merge dbSNP data with Sanger resequencing data)

Exceptions:

Those cases where SNPs are shown as part of a sequence

12 of 25

A missense SNP, C1858T, in PTPN22 (Tyrosine-proteinphosphatase non-receptor type 22) has been identified as agenetic risk factor for rheumatoid arthritis.This SNP is also referred to as R620W.

1. Find the SNPView page for this SNP.

2. Why are the alleles on this page given as A/G?

3. What is the minor allele of this SNP in Caucasians?

5 MINUTE EXERCISE5 MINUTE EXERCISE

13 of 25

SNPs in EnsemblSNPs in EnsemblGeneSNPView (1)

SNP alleles

Transcript

InterPro domains

14 of 25

SNPs in EnsemblSNPs in EnsemblGeneSNPView (2)

15 of 25

SNPs in EnsemblSNPs in EnsemblTranscriptSNPView (1)

Shows SNP alleles in different:

• Individuals (human):Celera HuAA, HuCC, HuDD and HuFF, Craig Venter, Jim Watson

• Strains (mouse, rat)

• Breeds (chicken, dog)

16 of 25

SNPs in EnsemblSNPs in EnsemblTranscriptSNPView (2)

Resequencingcoverage

Alleles indifferent individuals

SNP alleles

Differentindividuals

17 of 25

SNPs in EnsemblSNPs in EnsemblTranscriptSNPView (3)

18 of 25

1. Find the TranscriptSNPView page for human PTPN22.

2. Do all individuals (HuAA, HuCC, HuDD, HuFF, Venter and Watson) have resequence coverage at the position of the C1858T (R620W) SNP?

3. Has any of the individuals a higher risk to get rheumatoid arthritis based on its genotype at this position?

4. Is there an individual that is heterozygote at this position?

5 MINUTE EXERCISE5 MINUTE EXERCISE

19 of 25

Haplotypes and Linkage Haplotypes and Linkage DisequilibriumDisequilibrium

A haplotype is a set of SNPs on a single chromatid that are statistically associated

Linkage disequilibrium describes a situation in which some combinations of SNP alleles occur more or less frequently in a population than would be expected from a random formation of haplotypes from alleles based on their frequencies

20 of 25

Measures of LDMeasures of LD

• D = P(AB) – P(A)P(B)• D ranges from – 0.25 to + 0.25

• D = 0 indicates linkage equilibrium

• dependent on allele frequencies, therefore of little use

• D’ = D / maximum possible value• D’ = 1 indicates perfect LD

• estimates of D’ strongly inflated in small samples

• r2 = D2 / P(A)P(B)P(a)P(b)• r2 = 1 indicates perfect LD

• measure of choice

21 of 25

Linkage DisequilibriumLinkage Disequilibrium

LDView

It is also possible to export SNP information for upload into the HaploView software tool

22 of 25

Linkage DisequilibriumLinkage DisequilibriumLDTableView

23 of 25

Retrieve all non-synonymous SNPs for the human CFTR gene using BioMart and export their id, genomic position, alleles and peptide shift(hint: which dataset should you start with?).

5 MINUTE EXERCISE5 MINUTE EXERCISE

24 of 25

DAS SourcesDAS Sources

For human, data from the following DAS Sources can bevisualised on ContigView:

• DGV and DGV loci:Structural variations from the Database of Genomic Variations (CNVs, InDels, inversions etc.)

• RedonCNV regions and RedonCNV loci:Copy number variations from Redon et al. paper

• SegDup Washu:Segmental Duplications, University of Washington

25 of 25

QQ&&AAQ U E S T I O N SQ U E S T I O N S

A N S W E R SA N S W E R S