1. Applications in Bioinformatics, Proteomics, and Genomics
SNPs (1) J. Gray (UT)
The realization that DNA differs from person to person much more than researchers had suspected, may transform medicine but could also threaten personal privacy.
Science Dec

2. Todays lecture
1: Understanding Genetic Variation 2. What are SNPs? 3. Why should we care about SNPs ? 4. SNP Discovery – The SNP Consortium/The International HapMap/The 1000 Genomes Project 5. Haplotypes and how chromosomal recombination gives rise to new Haplotypes
6. Overview of SNP detection methods

3. 1: Understanding Human Genetic Variation

4. “Every drop of human blood contains a history book written in the language of our genes” - Spencer Wells
“The Journey of Man: A Genetic Odyssey” 2002

5. Founder mutations
Two men born in the US - thousands of miles apart - have a propensity to absorb iron so well that it can cause organ damage - a condition known as hereditary hemochromatosis. The error in their genes originated in a single European ancestor, whose ancestors now number nearly 22 million including the two men above (who might be surprised to know they are related).
The original mutation is known as a “founder mutation”. The study of these mutations is intimately linked to the study of the recent evolution and spread of the human species.

6. Human Migration in the past 100K
“Restless Genes”
National Geographic Jan 2013
“Once modern humans began their migration out of Africa about 60,000 years ago, they kept going until they had spread to all corners of the globe. How far and fast they went depended on climate, the pressures of population and the invention of boats and other technologies. Less tangible qualities also sped their footsteps,imagaination, adaptability and curiosity.

7. All humans are very closely related
Humans went through a very narrow genetic bottleneck - estimated only about 1 to 10 million humans in the world after the last ice age (10 k)

8. Human demographic history has shaped the pattern of variation observed in modern populations.
The general concensus is that Africa is the cradle of modern humans (approx 200k years ago)
Genetic data shows the ALL non-Africans are the descendants of a small group of Africans that moved into the middle east about 70K yrs ago.

9. See also http://www.bradshawfoundation.com/stephenoppenheimer/
The greatest diversity of genetic markers is in Africa indicating it was the earliest home of modern humans. Only a handful of people - carrying a few markers - left Africa seeding the genetic makeup of the rest of the world.
National Geographic March 2006
See also

10. Genetic mutations that act as markers, trace the journey of human migration. The earliest known mutation to spread outside of Africa is M168 (about 50 K yrs ago)
This graphic shows the Y chromosome of a Native American man with various mutations including M168, proving his African ancestry.

11. Founder mutations on Y chromosome give rise to Haplotypes
“Eurasian Adam”
In human genetics, Haplogroup CT is a Y-chromosome haplogroup, defining one of the major lines of common ancestry of humanity along father-to-son male lines.
Men within this haplogroup have Y chromosomes with the SNP mutation M168, along with P9.1 and M294. These mutations are present in all modern human male lines except A and B, which are both found almost exclusively in Africa.

12. Haplogroups Mutations
Y-DNA Haplogroup Mutations Table
Haplogroups Mutations
A   no mutations
B  SRY
C   SRY >M168
D   SRY >M168>M174
E   SRY >M168>M96
F  SRY >M168>M89
G   SRY >M168>M89>M201
H  SRY >M168>M89>M69
I   SRY >M168>M89>M170
J   SRY >M168>M89>M304
K   SRY >M168>M89>M9
L   SRY >M168>M89>M9>M11
M  SRY >M168>M89>M9>M5
N  SRY >M168>M89>M9>M214
O  SRY >M168>M89>M9>M214>M175
O3 SRY >M168>M89>M9>M214>M175>M122
P  SRY >M168>M89>M9>M45
Q  SRY >M168>M89>M9>M45>P36
R  SRY >M168>M89>M9>M45>M207
R1b SRY >M168>M89>M9>M45>M207>M343
The Y haplotype is very stable because there is no recombination happening with any other chromosome.
The mitochondrial genome supplies a similar grouping in the maternal lineage.

13. The pattern of genetic diversity in modern human populations, is the result of many evolutionary processes.
New tools/resources promise to help identify functional mutations important for normal phenotypic variation as well as susceptibility to genetic disease.
The same approaches are just as important for deciding how to protect biodiversity and in aiding plant breeding and animal husbandry

14. Q: How much do humans differ ?
A: very very very little! But everyone is unique
Human genome project involved DNA from 9 individuals from diverse ethnic backgrounds
Identified about 26,000-40,000 genes
Also revealed about 1.5 million Single Nucleotide Polymorphisms – SNPs (Snips)
These are the most prevalent form of genetic variation in humans – it was estimated there are 20 million SNPs (about 0.6% of the total genome)

15. (Single Nucleotide Polymorphism)
2: What is a SNP ?
(Single Nucleotide Polymorphism)

16. GCATGCATGCATGCAT |||||||||||||||| CGTACGTACGTACGTA GCATGCAaGCATGCAT
2: So what is a SNP ?
Gene allele A1
GCATGCATGCATGCAT
||||||||||||||||
CGTACGTACGTACGTA
GCATGCAaGCATGCAT
||||||||||||||||
CGTACGTtCGTACGTA
Gene allele A2
Comparing DNA between two individuals shows that about every 1.5 kb there is one base pair difference – a single nucleotide polymorphism (SNP).

17. When a variant nucleotide is present in more than one percent of a population, that DNA position is the location of the SNP. (less than 1% considered “rare” alleles).
Only 2% of genome encodes protein 93% of all annotated genes have 1 SNP 59% have 5 or more SNPs 39% have 10 or more SNPs

18. Often scientists distinguish between ancient “founder mutations” where surrounding DNA is same as others in the population and “hot spot mutations” which occur in error prone regions.
Sci. Amer. Oct 2005

19. Old Originals versus numerous newcomers Sickle cell anemia is most often caused by a “founder mutation” Achondroplasia (a form of human dwarfism) ordinarily results from a “hotspot mutation”
Sci. Amer. Oct 2005

20. Noteworthy Founder Mutations
Gene Condition Mutation origin Migration Possible Advantage
of 1 copy
HFE Iron overload NW Europe Across Europe Protection from
anemia
CFTR Cystic fibrosis SW Europe Across Europe Protection from
diarrhea
HbS Sickle cell Africa To New World Protection from
disease Middle East malaria
ALDH2 Alcohol Far east Asia North & West Protection from
toxicity across Asia alcoholism
LCT Lactose Asia West & North Allows animal milk
tolerance across Eurasia consumption
GJB2 Deafness Middle East West & North Unknown
across Europe
FV Blood clots W. Europe Worldwide Protection from
Leiden sepsis
Sci. Amer. Oct 2005

21. In addition to SNPs there are Copy Number variations (CNVs)
CNVs can be caused by structural rearrangements of the genome such as deletions, duplications, inversions, and translocations. Some associated with disease, most are not and some are advantageous
Approximately 0.4% of the genome of unrelated people typically differ with respect to copy number
This gene duplication has created a copy-number variation. The chromosome now has two copies of this section of DNA, rather than one.

22. 3: Why should we care about SNPs ?

23. 3: Why should we care about SNPs ?
We want to know the basis of human variation and disease susceptibility How can some who never smoke get lung cancer and others who smoke heavily stay cancer free ? Why do some people exposed to HIV never develop AIDS ?

24. 4: Study human evolution
SNPs are useful to : DNA fingerprinting for criminal or parental identification 2: Help map polygenic/disease traits by comparing DNA of groups with and without inheritance of that disease
3: Genotype-specific medication (pharmacogenomics)
4: Study human evolution

25. 4: SNP Discovery

26. 4: SNP Discovery
The urgency and importance of identifying thousands of SNPs resulted in 11 major pharmaceutical and technology companies cooperating and one large scientific trust to underwrite the work – the TSC.
Also see

27. Example of SNP Discovery
The Whitehead Institute –isolated DNA from 10 ethnically diverse humans (Pilot phase).
NA Ameridian Female
NA Melanese Female
NA Biaka Pygmy Male
NA08779A American Black Female
NA Chinese Male 
NA Japanese Female 
NA Russian Male
NA CEPH/Amish Female
NA CEPH/French Male
NA CEPH/Utah Female 

28. Example of SNP Discovery
First a pool of 24 DNAs was digested with one of several restriction enzymes, size fractionated and cloned into M13-based vectors.
Individual clones sequenced, repeats discarded, gene pairs accepted only if 99% homologous.
SNPfinder algorithm used to find base pair discrepancies, repeated clusters removed
Validations of SNPs – using Phred scores 20-51
Validation in 8 individuals – PCR and sequence candidate SNP regions – reject SNP if heterozygous in all individuals (assumed to be in a repeat region).
Isolated more than 1.5 million SNPs

29. The information is freely available.
See also
The Goal of the International HapMap Project is to develop a “haplotype” map of the human genome, the HapMap, which will describe the common (not rare) patterns of human DNA sequence variation (variants in >1% of population).
The HapMap has become a key resource for researchers to use to find genes affecting health, disease, and responses to drugs and environmental factors. Phase 3 has been completed and there >6 million SNPs defined.
The information is freely available.
(see Nature 27 Oct 2005 for report on phase 1 of project, Nature 18 Oct 2007 for phase II and 2 Sep 2010 for phase III)

30. Sequencing Entire Genomes – The Terabyte era
July 10, 2008
DNA sequencing enters the terabase era
The Wellcome Trust Sanger Institute announced something remarkable: its scientists had sequenced 300 human genomes in six months.
In perspective. They sequenced more DNA every 2 seconds than was sequenced during the first five years of international genome-sequencing efforts, from 1982 to The institute has now sequenced 1 trillion = 1000 billion letters of the genetic code.
The cost of sequencing a human genome has fallen from $3 billion in 2001 (Human Genome Project)
$1 million in 2007 (for James Watson)
$50,000 in 2010 (James Lupski)
Expect ~$1000 in 2013 (NIH goal)
-Get used to it!

31. Oxford Technolgies MiniION USB sequencer claims a $1000 genome
Technology is not commercial yet
High error rate
Cannot provide a full genome (yet)
Need to know Diploid genome
Comments on limitations

33. SNP Discovery by sequencing individual genome
Lupski, J.R. et al., New England Journal of Medicine 362:
James Lupski, a physician-scientist who suffers from a neurological disorder called Charcot-Marie-Tooth, searched for the genetic cause for > 25 years……..
Late last year, he finally found it-by sequencing his entire genome -in SH3TC2 (the SH3 domain and tetratricopeptide repeats 2 gene) – cost ~$50,000
First to show how whole-genome sequencing can be used to identify the genetic cause of an individual's disease.
"I have hundreds of thousands of differences from all the other genomes that have been sequenced. I expect that to hold true for others. Everyone is truly unique.”

34. SNP Discovery by sequencing family genomes
How much genetic variation in each family?
Sequenced entire genome of two parents and 2 children who both have a recessive genetic disease named Miller Syndrome
Estimated a human intergeneration mutation rate of ~1.1 x 10-8 per position per haploid genome
a high degree of certainty that each parent passes 30 new mutations—for a total of 60—to their offspring
Also narrowed candidate genes to just four
Roach et al., Analysis of Genetic Inheritance in a Family Quartet by Whole-Genome Sequencing.
Science DOI: /science March 2010

35. SNP Discovery by sequencing 1000 genomes
With advances in sequencing technology, the 1000 genomes project became feasible – revealed more SNPs than the HapMap project.
- useful video tutorials

36. Whose 1000 genomes?
Deep whole-genome sequencing of trios (mother-father-daughter) from 2 populations
Low-coverage sequencing of 179 unrelated individuals from 4 populations
Exon sequencing of 906 randomly-selected genes in 697 individuals from 7 populations.
Yielded 4.9 terabases of sequence!
15 million SNPs
1 million Indels (Insertions/Deletions)
20,000 structural variantsp

37. SNP Discovery by sequencing 1000 genomes
All data is deposited at 1000genomes.org
Paper: A map of human variation from population-scale sequencing Nature Vol 467 p 1061 October 2010

38. What sequencing 1000 genomes reveals
Variation is not evenly distributed in the genome (higher in telomeres, lower in gene dense regions
Diversity in exons is half that of introns
Most SNPs were already known of which 56% were present in all population panels, 25% in a single panel
Of new SNPS (novel variants) 4% were found in all panels and 84% in only one (more rare variants)
New germline mutations = about 1 in 10-8
68,300 novel non-synonymous variants
About Loss-of-function variants per individual, affecting genes (we are all mutants!)
Any individual genome differs by about 10,000 non-synonymous variants from the ref sequence
Culture cell lines accumulated hundreds of mutations not present in the germline

39. Would you have your genome sequenced if you could afford it?
Yes 81%
No 9%
Undecided 10%
If you had your genome sequenced would you want to know everything?
Yes 74%
No 16%
Undecided 10%
In 2013 Researchers were able to identify 50 people whose DNA had been posted anonymously on the Internet for genetics studies.
The results highlight a trade-off in making genetic data widely available for researchers and protecting personal privacy.

40. 5: Haplotypes and how chromosomal recombination gives rise to new Haplotypes
xyz
Xyz
Xyz
xYz
xYZ
XYZ
xyz
During meiosis, homologous chromosomes (1 from each parent) pair along their lengths. The chromosomes cross over at points called chiasma. At each chiasma, the chromosomes break and rejoin, trading some of their genes. This recombination results in genetic variation (new haplotypes).

41. Crossing over occurs during Meiosis
Crossing Over during Meiosis increases genetic variability
If every homologous pair in humans has just one crossing over event then there will many possible new gametes (sperm or eggs) with many new haplotypes (depends on how the chromosomes randomly segregate and how many).

42. SNP1 SNP2 C A C A SNP1 SNP2 SNP1’ SNP2’ T G T G SNP1’ SNP2’ Patient A
SNPs that are inherited close to one another on a given chromosome are said to be genetically “linked”
SNP1
SNP2
Maternal chromosome C A
Patient A C A
Paternal chromosome
SNP1
SNP2
SNP1’
SNP2’
Maternal chromosome T G
Patient B T G
Paternal chromosome
SNP1’
SNP2’

43. A Trio is the genotype of mother father and offspring
Haplotype refers to the set of alleles on one particular chromosome Patient C has two haplotypes
SNP1
SNP2 C A
Maternal chromosome
Patient C T G
Paternal chromosome
SNP1’
SNP2’
Each haplotype is passed on to offspring as a complete unit unless recombination occurs between them to create new haplotypes
A Trio is the genotype of mother father and offspring

44. SNP1 SNP2 C A T G SNP1’ SNP2’ SNP1 SNP2’ C G T A SNP1’ SNP2
Recombination in patient C leads to 2 new haplotypes in gametes (sperm or egg) that are passed onto next generation
SNP1
SNP2 C A
Maternal chromosome
Patient C T G
Paternal chromosome
SNP1’
SNP2’
SNP1
SNP2’ C G
“New” chromosome T A
“New” chromosome
SNP1’
SNP2

45. Because of recombination a haplotype that surrounds a founder mutation will get shorter over generations as chromosomes mix
Sci. Amer. Oct 2005

46. It follows that a “recent” founder mutation will be associated with a long haplotype, and an “ancient” founder mutation with a short haplotype.
Sci. Amer. Oct 2005

47. 6: How to detect SNPs ?

48. SNP assay requirements
a: Assay must be easily developed from sequence information b: Low cost of assay development (reagents/personnel) c: Assay must be robust d: Easily automated e: Simple analysis, accurate genotype calling f: Scalable assay (up to millions/day) g: Low cost per genotype assay
Genotyping methods are evolving rapidly and costs greatly decreasing

49. How can we detect SNPs ?
Since most association studies require genotyping large numbers of individuals with a large number of SNPs then SNP assays must clearly distinguish between different alleles. there are several methods and this is an area of intense investigation and improvement…………

50. Sequence-specific SNP Detection Methods
1: Hybridization: Allele-specific probes that only hybridize when there is a perfect match - several methods to detect hybridization

51. Affymetrix® SNP Array 6.0 1.8 million SNPs ~ $400

52. Sequence-specific SNP Detection Methods
2: Nucleotide incorporation: addition of nucleotides with DNA polymerase can only occur if 3’ end of primer is a perfect match with SNP

53. Now Infinium HD does up to 1.2 million
This method can be miniaturized and large numbers of SNPs assayed in a short time
e.g. Illumina Infinium II Assay Protocol - can assay 650,000 SNPs on one chip - three day protocol from start to finish
Now Infinium HD does up to 1.2 million
Illumina Omni 5 million SNPs $580
For online video see

55. Next lecture
1: Mapping complex traits using SNPs
2: Genome Wide Association Studies (GWAS)
3. Example of complex trait mapping Using SNP analysis to find gene linked to retinal dystrophy