![Page 1: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/1.jpg)
SNPs, Haplotypes,DiseaseAssociations
Algorithmic Foundations of Computational Biology II
Course 1
Prof. Sorin Istrail
![Page 2: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/2.jpg)
SNPs and the Human Genome:The Minimal Informative Subset
![Page 3: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/3.jpg)
Overview
Introduction:
SNPs, Haplotypes A Data Compression Problem:
The Minimum Informative Subset A New Measure:
Informativeness
![Page 4: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/4.jpg)
A Most Challenging Problem
“None of the [advances of the 20th century medicine] depend on a deep knowledge of cellular processes or on any discoveries of molecular biology.
Cancer is still treated by gross physical and chemical assaults on the offending tissue.
Cardiovascular Disease is treated by surgery whose anatomical bases go back to the 19th century …Of course, intimate knowledge of the living cell and of basic molecular processes may be usefuleventually.”
Lewontin (1991)
![Page 5: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/5.jpg)
Now
“A decade later, molecular biology can claim very few successes for drugs in clinical use that were designed ab initioto control a specific component of a pathwaylinked to disease: these include themonoclonal antibody Herceptin, and the kinase inhibitor Gleevec.”
Reik, Gregory and Urnov (2002)
![Page 6: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/6.jpg)
Introduction
SNPs, HAPLOTYPES
![Page 7: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/7.jpg)
A SNP is a position in a genome at which two or more different bases occur in the population, each with a frequency >1%.
GATTTAGATCGCGATAGAGGATTTAGATCTCGATAGAG
The most abundant type of polymorphism
The two alleles at the site are G and T
Single Nucleotide Polymorphism (SNP)
![Page 8: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/8.jpg)
tttctccatttgtcgtgacacctttgttgacaccttcatttctgcattctcaattctatttcactggtctatggcagagaacacaaaatatggccagtggcctaaatccagcctactaccttttttttttttttgtaacattttactaacatagccattcccatgtgtttccatgtgtctgggctgcttttgcactctaatggcagagttaagaaattgtagcagagaccacaatgcctcaaatatttactctacagccctttataaaaacagtgtgccaactcctgatttatgaacttatcattatgtcaataccatactgtctttattactgtagttttataagtcatgacatcagataatgtaaatcctccaactttgtttttaatcaaaagtgttttggccatcctagatatactttgtattgccacataaatttgaagatcagcctgtcagtgtctacaaaatagcatgctaggattttgatagggattgtgtagaatctatagattaattagaggagaatgactatcttgacaatactgctgcccctctgtattcgtgggggattggttccacaacaacacccaccccccactcggcaacccctgaaacccccacatcccccagcttttttcccctgctaccaaaatccatggatgctcaagtccatataaaatgccatactatttgcatataacctctgcaatcctcccctatagtttagatcatctctagattacttataatactaataaaatctaaatgctatgtaaatagttgctatactgtgttgagggttttttgttttgttttgttttatttgtttgtttgtttgtattttaagagatggtgtcttgctttgttgcccaggctggagtgcagtggtgagatcatagcttactgcagcctcaaactcctggactcaaacagtcctcccacctcagcctcccaaagtgctgggatacaggtgtgacccactgtgcccagttattattttttatttgtattattttactgttgtattatttttaattattttttctgaatattttccatctatagttggttgaatcatggatgtggaacaggcaaatatggagggctaactgtattgcatcttccagttcatgagtatgcagtctctctgtttatttaaagttttagtttttctcaaccatgtttacttttcagtatacaagactttgacgttttttgttaaatgtatttgtaagtattttattatttgtgatgttatttaaaaagaaattgttgactgggcacagtggctcacgcctgtaatcccagcactttgggaggctgaggcgggcagatcacgaggtcaggagatcaagaccatcctggctaacatggtaaaaccccgtctctactaaaaatagaaaaaaattagccaggcgtggtggcgagtgcctgtagtcccagctactcgggaggctgaggcaggagaatggtgtgaacctgggaggcggagcttgcagtgagctgagatcgtgccactgcattccagcctgcgtgacagagcgagactctgtcaaaaaaataaataaaatttaaaaaaagaagaagaaattattttcttaatttcattttcaggttttttatttatttctactatatggatacatgattgatttttgtatattgatcatgtatcctgcaaactagctaacatagtttattatttctctttttttgtggattttaaaggattttctacatagataaataaacacacataaacagttttacttctttcttttcaacctagactggatgcattttttgtttttgtttgtttgtttgctttttaacttgctgcagtgactagagaatgtattgaagaatatattgttgaacaaaagcagtgagagtggacatccctgctttccccctgattttagggggaatgttttcagtctttcactatttaatatgattttagctataggtttatcctagatccctgttatcatgttgaggaaattcccttctatttctagtttgttgagattttttaattcatgtgattgcgctatctggctttgctctca
tc
ga
ga
ga
ga
ga
gc
gc
gc
tc
ga
ga
ga
ga
ga
tc
tc
tc
tc
ga
ga
ga
tc
gc
tc
tc
tc
Human Genome contains ~ 3 G basepairs arranged in 46 chromosomes.
Two individuals are 99.9% the same. I.e. differ in ~ 3 M basepairs.
SNPs occur once every ~600 bp
Average gene in the human
genome spans ~27Kb
~50 SNPs per gene
![Page 9: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/9.jpg)
G C T C G A C A A C A GG T T C G T C A A C A G
Two individuals
C A G HaplotypesT T G
SNP SNP SNP
Haplotype
![Page 10: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/10.jpg)
Mutations
Infinite Sites Assumption:
Each site mutates at most once
![Page 11: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/11.jpg)
Haplotype Pattern
0 0 0 01 1 0 10 0 1 00 1 0 1
C A G TT T G AC A T GC T G T
At each SNP site label the two alleles as 0 and 1.
The choice which allele is 0 and which one is 1
is arbitrary.
![Page 12: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/12.jpg)
G T T C G A C T A T T A
G T T C G A C A A C A TA C G T A T C T A T T A
Recombination
![Page 13: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/13.jpg)
G T T C G A C T A T T A
G T T C G A C A A C A TA C G T A T C T A T T A
The two alleles are linked, I.e., they are “traveling together”
?
Recombinationdisrupts the linkage
Recombination
![Page 14: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/14.jpg)
Variations in Chromosomes Within a Population
Common Ancestor
Emergence of Variations Over Time
time present
Disease Mutation
Linkage Disequilibrium (LD)
![Page 15: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/15.jpg)
Time = present
2,000 gens. ago
Disease-Causing Mutation
1,000 gens. ago
Extent of Linkage Disequilibrium
![Page 16: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/16.jpg)
A Data Compression Problem
The Minimum Informative Subset
![Page 17: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/17.jpg)
A Data Compression Problem Select SNPs to use in an association study
Would like to associate single nucleotide polymorphisms (SNPs) with disease.
Very large number of candidate SNPs Chromosome wide studies, whole genome-scans For cost effectiveness, select only a subset.
Closely spaced SNPs are highly correlated It is less likely that there has been a recombination between two
SNPs if they are close to each other.
![Page 18: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/18.jpg)
Disease Associations
![Page 19: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/19.jpg)
Association studies
DiseaseResponder
ControlNon-responder
Allele 0 Allele 1
Marker A is associated with
Phenotype
Marker A:
Allele 0 =
Allele 1 =
![Page 20: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/20.jpg)
Evaluate whether nucleotide polymorphisms associate with phenotype
T A GA A
C G GA A
C G TA A
T A TC G
T G TA G
T G GA G
Association studies
![Page 21: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/21.jpg)
T A GA A
C G GA A
C G TA A
T A TC G
T G TA G
T G GA G
Association studies
![Page 22: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/22.jpg)
1 1 00 0
0 0 00 0
0 0 10 0
1 1 11 1
1 0 10 1
1 0 00 1
Association studies
![Page 23: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/23.jpg)
Compression based on Haplotype Resolution
![Page 24: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/24.jpg)
0 1 01 1
1 0 00
0 0 10 1
1
For a SNP s we associate a bipaprtite graph.
Nodes: the set of haplotypes.
Edges: the set of pairs of haplotypes with different alleles at s.
s1
s2
D-graph of a SNP
![Page 25: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/25.jpg)
0 1 01 1
1 0 00
0 0 10 1
1
For a set of SNPs S we associate a bipaprtite graph.
Nodes: the set of haplotypes.
Edges: the set of pairs of haplotypes with different
alleles at some SNP s in S.
s1
s2
D-graph of a set of SNPs
![Page 26: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/26.jpg)
0 1 01 1
1 0 00
0 0 10 1
1
Red SNP is equivalent to Blue SNP
SNP Selection
![Page 27: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/27.jpg)
Red SNPs predict Green SNP
0 1 01 1
1 0 00
0 0 10 1
1
SNP Selection
![Page 28: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/28.jpg)
Minimal Informative Subset
0 1 01 1
1 0 00
0 0 10 1
1
Data Compression
![Page 29: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/29.jpg)
Compresssion based on Haplotype Blocks
![Page 30: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/30.jpg)
Hypothesis – Haplotype Blocks?
The genome consists largely of blocks of
common SNPs with relatively little recombination
within the blocks Patil et al., Science, 2001; Jeffreys et al., Nature Genetics, 2001; Daly et al., Nature Genetics, 2001
![Page 31: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/31.jpg)
Sense genes
Antisense genes
200 kb
1 2 3 4
DNA
SNPs
Haplotypeblocks
Haplotype Block StructureLD-Blocks, and 4-Gamete Test Blocks
![Page 32: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/32.jpg)
Hudson and Kaplan 1985
A segment of SNPs is a block if between every pair of SNPs at most 3 out of the 4 gametes (00, 01,10,11) are observed.
0 0 10 1 11 1 01 1 1
0 0 10 1 11 1 01 0 1
BLOCK VIOLATES THE BLOCK DEFINITION
Four Gamete Block Test
![Page 33: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/33.jpg)
Finding Recombination Hotspots:Many Possible Partitions into Blocks
A C T A G A T A G C C TG T T C G A C A A C A TA C T C T A T G A T C GG T T A T A C G A C A TA C T C T A T A G T A TA C T A G C T G G C A T
All four gametes are present:
![Page 34: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/34.jpg)
A C T A G A T A G C C TG T T C G A C A A C A TA C T C T A T G A T C GG T T A T A C G A C A TA C T C T A T A G T A TA C T A G C T G G C A T
Find the left-most right endpoint of any constraint and mark the site
before it a recombination site.
Eliminate any constraints crossing that site.
Repeat until all constraints are gone.
The final result is a minimum-size set of sites crossing all constraints.
![Page 35: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/35.jpg)
Data Compression
ACGATCGATCATGAT
GGTGATTGCATCGAT
ACGATCGGGCTTCCG
ACGATCGGCATCCCG
GGTGATTATCATGAT
A------A---TG--
G------G---CG--
A------G---TC--
A------G---CC--
G------A---TG--
Haplotype Blocks based on LD(Method of Gabriel et al.2002)
Selecting Tagging SNPs in blocks
![Page 36: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/36.jpg)
A New Measure
Informativeness
![Page 37: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/37.jpg)
Informativeness
0 1 00 1
0 1 10 0
s
h2
h1
![Page 38: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/38.jpg)
1 0 00 0
0 1 00 1
0 1 10 0
1 0 11 1
s1 s2 s3 s4 s5
I(s1,s2) = 2/4 = 1/2
Informativeness
![Page 39: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/39.jpg)
1 0 00 0
0 1 00 1
0 1 10 0
1 0 11 1
s1 s2 s3 s4 s5
I({s1,s2}, s4) = 3/4
Informativeness
![Page 40: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/40.jpg)
1 0 00 0
0 1 00 1
0 1 10 0
1 0 11 1
s1 s2 s3 s4 s5
I({s3,s4},{s1,s2,s5}) = 3
S={s3,s4} is a
Minimal Informative Subset
Informativeness
![Page 41: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/41.jpg)
Minimum Set Cover= Minimum Informative Subset
s1
s2
s5
s3
s4
e1
e2
e3
e4
e5
e6
SNPs Edges
1 0 00 0
0 1 00 1
0 1 10 0
1 0 11 1
s1
s2
s3
s4
s5
Graph theory insight
Informativeness
![Page 42: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/42.jpg)
Minimum Set Cover {s3, s4}= Minimum Informative Subset
s1
s2
s5
s3
s4
e1
e2
e3
e4
e5
e6
SNPs Edges
1 0 00 0
0 1 00 1
0 1 10 0
1 0 11 1
s1
s2
s3
s4
s5
Informativeness
Graph theory insight
![Page 43: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/43.jpg)
Real Haplotype Data
Two different runs of the Gabriel el al Block Detection method +
Zhang et al SNP selection algorithm
Our block-free algorithm
A region of Chr. 22
45 Caucasian samples
![Page 44: SNPs, Haplotypes, Disease Associations Algorithmic Foundations of Computational Biology II Course 1 Prof. Sorin Istrail](https://reader035.vdocuments.net/reader035/viewer/2022062409/5697bfd91a28abf838cafb8e/html5/thumbnails/44.jpg)
When Maximum Likelihood = Bayesian = Parsimony
A C G T
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
101112131415
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789101112131415
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
123456789
1011121314
1 2 3 4 56 7 8 9101112131415161718192021222324252627282930
1234567891011121314