Download SNP Discovery by sequencing 1000 genomes

Applications in Bioinformatics,
Proteomics, and Genomics
SNPs (1)
J. Gray (UT)
[email protected]
Science Dec 21 2007
The realization that DNA differs
from person to person much more than
researchers had suspected, may
transform medicine but could also
threaten personal privacy.
www.sciencemag.org/sciext/btoy2007
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
1:
Understanding
Human Genetic
Variation
“ 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
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.
“Restless Genes”
National Geographic Jan 2013
Human Migration
in the past 100K
https://genographic.nationalgeographic.com
“ 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.
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)
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.
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 http://www.bradshawfoundation.com/stephenoppenheimer/
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.
Founder mutations on Y chromosome
give rise to Haplotypes
“Eurasian Adam”
In human genetics,
Haplogroup CT is a Ychromosome haplogroup,
defining one of the major
lines of common ancestry
of humanity along fatherto-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.
Y-DNA
Haplogroup
Mutations
Table
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.
Haplogroups Mutations
A no mutations
B SRY10831.1
C SRY10831.1>M168
D SRY10831.1>M168>M174
E SRY10831.1>M168>M96
F SRY10831.1>M168>M89
G SRY10831.1>M168>M89>M201
H SRY10831.1>M168>M89>M69
I SRY10831.1>M168>M89>M170
J SRY10831.1>M168>M89>M304
K SRY10831.1>M168>M89>M9
L SRY10831.1>M168>M89>M9>M11
M SRY10831.1>M168>M89>M9>M5
N SRY10831.1>M168>M89>M9>M214
O SRY10831.1>M168>M89>M9>M214>M175
O3 SRY10831.1>M168>M89>M9>M214>M175>M122
P SRY10831.1>M168>M89>M9>M45
Q SRY10831.1>M168>M89>M9>M45>P36
R SRY10831.1>M168>M89>M9>M45>M207
R1b SRY10831.1>M168>M89>M9>M45>M207>M343
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
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)
2: What is a SNP ?
(Single Nucleotide Polymorphism)
2: So what is a SNP ?
GCATGCATGCATGCAT
||||||||||||||||
CGTACGTACGTACGTA
GCATGCAaGCATGCAT
||||||||||||||||
CGTACGTtCGTACGTA
Gene allele
A1
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).
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
93%
59%
39%
2% of genome encodes protein
of all annotated genes have 1 SNP
have 5 or more SNPs
have 10 or more SNPs
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
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
Noteworthy Founder Mutations
Gene
Condition
Mutation origin
HFE
Iron overload
Possible Advantage
of 1 copy
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
NW Europe
Migration
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.
3: Why should we care about
SNPs ?
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 ?
SNPs are useful to.......
1: 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
4: SNP Discovery
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 http://www.wtccc.org.uk
Example of SNP Discovery
The Whitehead Institute –isolated DNA from
10 ethnically diverse humans (Pilot phase).
NA10965
NA10540
NA10470
NA08779A
NA11322
NA11589
NA13820
NA13117
NA12615
NA11997
Ameridian Female
Melanese Female
Biaka Pygmy Male
American Black Female
Chinese Male
Japanese Female
Russian Male
CEPH/Amish Female
CEPH/French Male
CEPH/Utah Female
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
www.hapmap.org
See also
http://www.ncbi.nlm.n
ih.gov/SNP/
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)
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 genomesequencing efforts, from 1982 to 1987. 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!
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
https://www.nanoporetech.com
Comments on limitations
http://www.facebook.com/notes/brandon-colby-md/a-physiciansthoughts-on-oxford-nanopores-minion-and-gridion-dnasequencing-devi/320675544646237
SNP Discovery by sequencing individual genome
Lupski, J.R. et al., New England Journal of Medicine 362:11811191 2010
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.”
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: 10.1126/science.1186802 March 2010
SNP Discovery by sequencing 1000 genomes
With advances in sequencing technology, the 1000
genomes project became feasible – revealed more SNPs
than the HapMap project.
www.genome.gov/27542240 - useful video tutorials
Whose 1000 genomes?
Deep whole-genome sequencing of trios (motherfather-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
SNP Discovery by sequencing 1000 genomes
http://browser.1000genomes.org/index/html
All data is deposited at 1000genomes.org
Paper: A map of human variation from population-scale sequencing
Nature Vol 467 p 1061 October 2010
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 340-400 Loss-of-function variants per individual,
affecting 250-300 genes (we are all mutants!)
Any individual genome differs by about 10,000 nonsynonymous variants from the ref sequence
Culture cell lines accumulated hundreds of mutations not
present in the germline
Would you have your genome sequenced if you
could afford it?
Yes
No
Undecided
81%
9%
10%
If you had your genome sequenced would you
want to know everything?
Yes
No
Undecided
74%
16%
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.
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).
Crossing over occurs during Meiosis
http://www.youtube.com/watch?v=BhJf9MHHmc4
http://www.youtube.com/watch?v=3qgBKrAZCLg
Crossing Over during Meiosis increases
genetic variability
http://www.dnatube.com/video/350/Crossi
ng-Over-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).
SNPs that are inherited close to one another
on a given chromosome are said to be
genetically “linked”
SNP1
C
Patient A
C
SNP1
SNP2
A
A
SNP2
SNP1’
T
Patient B
T
SNP1’
SNP2’
G
G
SNP2’
Maternal
chromosome
Paternal
chromosome
Maternal
chromosome
Paternal
chromosome
Haplotype refers to the set of alleles
on
one
particular
chromosome
Patient C has two haplotypes
SNP1
C
Patient C
T
SNP1’
SNP2
A
G
SNP2’
Maternal
chromosome
Paternal
chromosome
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
Recombination in patient C leads to 2 new
haplotypes in gametes (sperm or egg) that
are passed onto next generation
SNP1
C
Patient C
T
SNP1’
SNP2
A
G
SNP2’
Maternal
chromosome
Paternal
chromosome
SNP1
C
T
SNP1’
SNP2’
G
A
SNP2
“New”
chromosome
“New”
chromosome
http://www.youtube.com/watch?v=3qgBKrAZCLg
Because of recombination a haplotype that
surrounds a founder mutation will get shorter
over generations as chromosomes mix
Sci. Amer. Oct 2005
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
6: How to detect SNPs ?
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
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…………
Sequence-specific SNP Detection Methods
1: Hybridization: Allele-specific probes that
only hybridize when there is a perfect match several methods to detect hybridization
Affymetrix® SNP Array 6.0
1.8 million SNPs ~ $400
http://www.affymetrix.com/estore/browse/staticHtmlContentTemplate.jsp?stati
cHtmlMediaId=m1621192&isHtmlStatic=true&navMode=35810&aId=productsNav
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
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
www.illumina.com
Illumina Omni 5 million SNPs $580
For online video see
http://www.illumina.com/applications/genot
yping.ilmn
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