summer 2005 show me the cpg islands! alicia laughton (mathematics ‘06) jessica minnier...

Summer 2005

Show Me the CpG Islands!

Alicia Laughton (Mathematics ‘06)

Jessica Minnier (Mathematics ‘07)

Guided by Yung-Pin Chen (Mathematics/Statistics)

(With Statistical Significance)

Summer 2005

This work is funded byJohn S. Rogers

Science Research Program

Summer 2005

Outline

• DNA Overview

• CpG Islands

• Methods– Traditional Method

– Our Method

• Future plans

Summer 2005

DNA

• Deoxyribonucleic acid

• Double-helix• Chain of nucleotide

subunits• Contains genetic

information

Summer 2005

Nucleotides

• Made up of sugar, Phosphate, and bases

• Four bases– Adenine (A)

– Cytosine (C)

– Guanine (G)

– Thymine (T)

• CpG represents a C directly followed by a G in the DNA sequence

Summer 2005

Methylation

• Causes C to turn into T• Accounts for low

occurrence of CpG dinucleotides in vertebrates– Expectation is 6.25%

randomly

– Actually 1% of total sequence (Bird 1986)

Summer 2005

Sequence AL031723

• Human DNA sequence on chromosome 16• 3 known CpG Islands• Percentage of Content:

– A - 22.7%

– C - 29.5%

– G - 28.3%

– T - 19.5%

– CpG - 3.1%

Summer 2005

CpG Islands

• “regions of DNA with a high G + C content and a high frequency of CpG dinucleotides relative to the bulk genome” -- Gardiner-Garden and Frommer (1987)

Summer 2005

CpG islands & Genes

Gene

5’ end

CpGi

Gene

Promoter CpG islands

Gene CpG islands in body

Gene 3’ end CpG islands

Summer 2005

What is important about CpG Islands?

• Useful in identifying protein-coding regions (Yoon and Vaidyanathan, 2004)

– Associated with “housekeeping genes” and 40% of tissue-specific genes

• Aberrant methylation of CpG sites may cause silencing of tumor-suppressor genes (Deng, Zhou et al, 2002)

Summer 2005

aggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagtgtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttgagacggagtcttgctctgtcacccaggctggagtgtagtggtgcaatctcggctcactgcacctctgcctcccgggttcaagcgattttcctgcctcagcccccggagtagctgggattacaggtgcccgccaccacacccagctaatttttgtatttttagtagagacggggtttcgccatgttggccaggctggtctcgaactcctgacctcaggtgatccgcctgcctcagcctcccaaagtgctgggattacagacgtgagccactgcggctggcctctctccccgtctttaactgtagccctgtgaattctcatcagcctgggcctggactcagcaggccaaaaagttaccagcagagcccagcacatgtgaggaaagtcggagacgtggcggcgccggccggaggatccttcccaagaccctgggccgctgtggccccctagatcttgcaggttgccagggtgccaggccagggagggggcctttctgagattctcctcattctgacacaggagaggagggcactgacccagtcccaaggtcccgggggaatcagccgaccacagcccaggactgtcccacctgggcagagagcccattctgggtgcccagcccgggcaggcccaggcacccccagcagtgccccgggcagcacctgccagccaggtagtgcagggtgaggttgggcagggcagggcgtggtaggtcagctgagcaaacagctcggagggagagctggggagggctgggaactaggtcgatagaaacacagggactgtgttagggaggggatgccttgccagtcacgcccagccctgactcctgccctctgagggggcttcccccacccctgctgacagccccaggaccggcccctgccaggaggctgacctgccaggagtgaccgccccagacttgagcccttgggaggcaggttctgagtccccttttcctgctcagacccccagggaaacgcaggctgggccagaggcagctgcacagacccctgcagtggggtgctcggtggagagcgctggaggtgggagggaggatgtgtgaggcagcgggagagaatccaggcttcccccacaacacccaccatgagcggtgcagagtaggggtgggcggcacgggagccttcccaccccgcagaaccaggccctgggcagagctggcctacagacgataccggacaagtcctcctccgtcttggtgacagagggagctgggactccctccacccacccactgccacttcagaagcagccacagggagactgggaggggcaggggtgctggggatgagcgtggggctcagccctccctcttcccaccctggagggctgcctccttccagcccacctggaagggtggtgtcagtcccagagcccctgcactccccgccccacctcctgcagctggaacccgcgtgggagccgcacccagcgtcccagggacaaacacagaggccttgggtggtggcggtaccaaggtctgaggcctggcagctcaggggcacccccgtccctgagagaggtcaagaaggggaggcaccaccccccaccacgggacctcgctgacgatgcccatagagagaaaccaggccagtgctgggaggggaaagaccccaggcctcatgagaagtcactgcctgcttttcccctcggccaggaaggaagccccaggcccttccctcccgtctcgggcatactgaccccaggcaccaagcgagaccaggagcccacccctttcctttcccagatggcacaccagtgactctgaatatcggagcgcacccctgctccctgggaggcaggatatcgtgccgctgctccctggggcgcacgataccctccccaggaaggcgccggtcagggcggacgggccagggtgctcaccggtaccaggcgaggccgcgctcgtagcacctgtcgaagaagtggggctcagagcccagcgcgcggacgtcggggtgcagccgcagaaactccagcagggcgcgcgtgccgcccttcttcacgccaacgatgagcgcttgcgggaagcgccggcggccgggaccgctggccaaaggcaggccgggtgctcccgggcggtggacggagctggacggctcggagggcgcgggggccggcgcgggggcgcgggcggccggcgggcagcggccggggagggcgcagaggcagtaggcgccgagcaccagggccacgagcagcatcggcgcgcgggacgcccgcagagcggccccttgcccggcccctgcgccctggccgcccccggccccgccgcccaggccgccgctacctgccatggggtcgcgccgctccaggcccgggagcgggggcagcaggcgggcgcgcatctcggcccgcgcgccgctcagtccgtgggtgcccggcttgtgctctgcgcccggcggtcccgcagcctgggagcgggcgcggggcgggaccgggggcggggtctggacgccctcccccctccccctcccccgcccactccgcctccgaggccactgcctgggctggacccgccggcagccgccaccacccgggcgcgactcgagctgccgggaccaccaggacgctcctgctccgagatcccaggccctggctcgcttgactccggcatcttcacctctgcgcggggaggatgcggcggcggtggccgttcgggacgcagggcagggacagggcggcgcgcgggcctcgggaccctctgtttgaagaccgatccccttccccccccaccccactccgggacgtgcgcggcaggtgcataggccaagccttggcctgcaggagcgggagcctcatcgccaggccaaggggacccaggaaaagcgtcgatccgggcactcggcctgccaagggagaaagaggccgggacagcaccctagtgtgcagagagggatcccagaacgtgtggggggagtctgcggccgggaatggcgtgcgcctcctcttcctgcctgctggagggaccagcaccaaaacaggaaagttcaccctgccaggccttctctccaaagagtcagagggagctccgtagggggatggggttcccggaccccctgccgtggaaggggagtgggaacacagacaggcggcaagggctttcgaggccccctcttgcacaaaccagctcagagatcggagatctttgggatcaattactttccctccccaggcatccgaagcctatcctagcccaggtgtggatgagggtgggagagacgggggaggagggagaggagcaggactggacccccgtgtgacaaacatctgacaagttgctctgaggactgcccccctccttgtggagcccacctcatctggtgtgcatttccctgcggctttcatccagccctgggcgaccctccctcctccatctcagcctccctcctcctgccccacacctcaggcctgggactcgcagatgccaaaagggcctggcagatgccaaagccagaaagtgcagggggactgcatcccccacaggagaccgggttcttccccactacatactcagaccccactccctgcacccactgctcttgcaaaccaggaactaaggggttcccctacccaccccgctccttgcctcctcttgcttttcttttgttttgtttgtttttgagacagagctgcactccagctgactcttgtcgcccaggctggagtgcagtggcacaatctcagctcactacaacctctgcctcccgggttcaagcgattctcctgcctcagcctcccaagtagctgggaatacaggcacccatcaccacgcctggctaatttttgtatttttagtagagatggggtttcaccatgttagtcaggctggtctcaaactcctgacctcaggtaatctgcccacctcagcctcccaaagggctgggattacaggcgtgagccactgtgccccaccctcctcttgcttttctaaaagatgatggtcaaagtacagcccccatttgcccccagacagggcacccttcccagatcgagaccttggggagtctgcgtgacccccacacctggcagacacaggtgcttcactagtgggggaacggctgagcatgtgctgagctcgggggcactagtgggctacagtccccaagtgggaggcccctcaagagcctggatgagctgactgacggtggagaggagggaaggagggcctatggccaaagtcaatccaggacccaactgccgaggccacaggaaggccgggtcaccgcctggaactaggtcggtcacagcccagtgggagccgtggcccggagactcaactgggggccctggttactctgctcgcctccccgcgtcggcacccagaacagagcttgcaggcactgggggcccagtccagggtctcaagagcagacaatgctgccttgcagttggggaaactgagacagggtgagaactttcagaggctcattgcaggctcctagcaggctgaaaggacggaggcacaggcacctaggagcacaccagccccacgtggccacggcccctcggagagcatgaggacacttgcaatgcggaagctcagcaggcccagctctactggctctgcaccgcccagtgaggggtcagcacagttggtccaagggacaataccagattaatgaggcagaagccacgggactgaccccttggaattctccacacccacactgtgcatccttaacccaaagcttctagcttggtagcccctcctaccctcctccctgcagcagggattagggatgcattctgacccctgcctgccgtcaggggagtgaggtctctccctggagcctgagctgaggatgcccaattcagccaggtgagccccgggatggactccatgtcccctagccaccacctgacttccccagcaccccacactggcaccagcccttcagatctcagaagcgagccaccctattctcacggagccccttcctgcctgccctccaaacccaagagtagttttagtacaaaaggcaaagttaacaaataggggtaggcgtcagggaaggaagaggatcagaggatcgggaacggagaaactggagcacctggagaagcgtctgggtcctgccacccccactgactccccaactggccttgggcagggtcctctctgcaggcgctgggtccaagcttggggatgagcagccaccagcgcgggctgcttcagctgaggctgccgcacccccacgtccatcctgggtagaggcaggacagccacagagccccatgcacggggctggactcaccctgggcactcacctaaaggcagtctcctcctttccaaagcccagactttctccggactcccaggaccaccaacaagggttcctgtgcgcagactcgggggtcttggggaggaaggacgctttctaggtggctgcctggaacctggaggcccctttctacagtacctggccagcggtcggtcacacctgagtgcccagagtgagcgggcggcagaggcatttctgacgctgccaggtaatcccacgggctggaaacgacctctgggctgggaagccaccgcctcccccagtcctgctgggtccctcagcagagagaacggaaccggggctttccccacagttttcaaagtttcagggaatcctagccaagtatcattccttcttccggagccgggaccccaggtcaagcctggggcccccacagggcggtcccaaccccactgcccggagcgcacccctgctccctgggaggcaggatatcgtgccgctgctccctggggcgcacgataccctccccaggaaggcgccggtcagggcggacgggccagggtgctcaccggtaccaggcgaggccgcgctcgtagcacctgtcgaagaagtggggctcagagcccagcgcgcggacgtcggggtgcagccgcagaaactccagcagggcgcgcgtgccgcccttcttcacgccaacgatgagcgcttgcgggaagcgccggcggccgggaccgctggcgtttccctcccaggggcccagtggtgaactgaattcaggcctgagacatactctgtctactaagtcaccccatctgcccagccttggtccacctggcactgcccagagacatcagtgatgcatttcggaagctggcaaagtggaccccactggagtacaaaggactcagggacccctgtgctggggaagagaaggagcccaggacctcccccaggggctgcctctgaggggcgtgagattcaggggcctctcgggtgggacctgcgggggccgctagacactgcgggaacttcacatccccaacgcccagcagcagcctgcagggaaggcaggggaggcgagccgggctcagagagggcgagcaacttgccccatccgaaggcaaaggtggtatgagacccgggtcctctctccacctctgccccagccttcctggccacagggctggcgccaggcaggcacggcacaggctcccggcagaggccacggtctcagccatccccacggtctcaggagtccccacggtctcagccgtccccacggtctgagtccccacggtctcagctgttcccacggtctcaggagtccccacaggttcagcagtccccacggtctcagccatccccacggtctcagccgtccccacagtctcagccatccccacggtctcagcagtccctactcaggacttgaaattccagcactggttccgtgatggctcctccagccccctgcccagcccagcatggtcatttccatctcctggcctttccgctgccgtctctctgctggatgctttatccttagtccccgctgagggcagaaggactttccaggaggaattgaccagaacgcagaacagcaggatgtggaatggactggggacagggagagagagatgcagggaccaggagtcggctcggagggttctcctggaagctgacccctccctccatcaggcactcggctgacggtggctacacacctcggggcgcccaggatggcagcactggggctgttcattcaccagtggatccccagcacctaacagagcctggcacgcagtggacattccattaatgtcgctcagtggaagggtatacgtgggaggagaggtcgggaaggctttctggaggtgacggccaggtgaagacgaggagaacagcattccaggccaaggaaccgtgtgggtgaaggctcagcagcagagagcccgggcagtagaggatggggtggagcttaaggccctgcgggaacaggggcggggcttagagtctggcctgaggctggtccagccccgcctcctcctcaggctcccaccaactctgagccaccagaccctcctttgtaaaatgaagacctcagtcatgactcgcatgagtctctgaagagtaacagctttattgtgatgtaattcacacaccactcaatccagccatttgtcgcatgcaaatcaatggttttcagtatattcatagtcgtgcaatcacaatcaattttagaacatttctatcaccccaaaaagaaatcctgtgtccattagcaatgacgccctcttctccccttcccacagcccctggcaaccacgaatctactttctgtctctatgggtttgcctattctggacatttcacaaaaagagaatcattgcttgaagccaggagttcaagaccaacctgggcaacaaagcgagaaccccgtctgtacaaaatattttaaatttagccaggcacagtggcgcacaccagtagtcccagcactttggaagtctgaggcaggaggttcacttgaggcggggaattcaaaaccagcctgggcaacatagggagtaccagtctctacaaaaaatttcaaaatttgccaagcgtgatggtatgcacctatagtcctagcttactcaggaggctgaggtgggaggatcgcttgagcccaggagtacgaggctgcagtgagccatgatcataccactgcattccagcctgggcgacagagtgagagcccatctctaaaacagaaagaaagaaagaaagaaatatggccagtcacagtggctcatgcctgtaatcccagcattttgggaggccaaggcaggtggatcacttgaggtcaggagttcgagaccagcctggccaacatggtgaaaccctgtctctaccaaaaatacataaattagccaggtgtgggccaggcgccatggcttacacttgtaatcccagcactttgggaggccgaggtgggcagatcacctgaggttgagagttcgagaccagcctgaccaacatgaagaaaccctgtctctactaaaaatacaaaaaattagctgggtgtggtggtgcatgcctgtaatctcagctacttgggaggctgaggaaggagaatggcttgaacccgggaggcagaggttgtggtgagccgagatcgcgcgattgcactccagcctgggcaacaacagcaaaactccatctcaaataataataataataaattagccaggtgtggtggtgcacgcctgtagtcccagctactcgggaggctgaggcacaagaaacccttgaacccgggaggcagaggttgcagtgaagctgaaattgcaccattccactccagcctgggagacagagtgagacaccatctctaaaatgaaaaaaaaaaaagagaatcatacaatgttcgtccttttgtgtctgggtctcttactcagcatgttctccaggttcatcaacactgtggcatgtgccagtacctccttcctcttcctgactgagtaatactccatcgtatggatggaccaccttttgttgattccctcattcgttgatggacatctaggttgtttccactgcggggttcttagtaacggtattacagggaaccatagattaccaggtatt

How do you locate the CpG island in a DNA sequence?

Summer 2005

agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctcaaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacgaggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtgggcgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgccactgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtgcccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaagctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccccagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgccggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttgaggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacctctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgctgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgggcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtccggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttcggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggaccaaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcggctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgctgctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtcctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccccgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagtgtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg

C+G Content: 0.492 Observed/Expected: 0.548

Summer 2005



Summer 2005


C+G Content: 0.712 Observed/Expected: 0.604 200 steps later…

Summer 2005


C+G Content: 0.598 Observed/Expected: 0.421 600 steps later…

Summer 2005

Just a couple formulas…

G+C content =

(# of C’s) + (# of G’s) length of window

Obs/Exp ratio =

Observed # of CpGs # of CpG’s in windowExpected # of CpGs (# of C’s)x(# of G’s) length

=

From window

Summer 2005

Traditional Methods• Gardiner-Garden and Frommer (1987)

– Window size 100 bp and Shift size 1bp– Criteria

• At least 200 base pairs• G + C content greater than 50%• Expected portion of the Obs/Exp ratio calculated over the window• Obs/Exp ratio greater than 0.6

• Takai and Jones (2002)– Window size 200 bp and Shift size 1bp– Criteria

• At least 500 base pairs• At least 7 CpG dinucleotides in 200 base pair sequence• G + C content greater than 55%• Obs/Exp ratio calculated in same fashion as above method• Obs/Exp ratio greater than 0.65

Summer 2005

The Traditional Method

C+G content Obs/Exp ratio

C+

G c

onte

nt/O

bs-E

x p r

ati o

Base Position

Sequence AL031723

Summer 2005

• Modifying the traditional methods

– Window size 200 bp and Shift size 1 bp

– Expected portion of the Obs/Exp ratio is based on whole sequence

• And….

Our Method

Observed # of CpGs # of CpG’s in windowExpected # of CpGs (# of C’s)x(# of G’s) length

=

From entire sequence

Summer 2005

• Cutoffs greater than 97th percentile of observed sequence

Obs/Exp Ratio G+C Content

Mean: 0.0018

Standard Deviation: 0.0014

97th percentile: 0.0058

Mean: 0.5815

Standard Deviation: 0.0818

97th percentile: 0.7350

G+C ContentObs/Exp Ratio

Num

ber

of O

bser

vati

ons

Num

ber

of O

bser

vati

ons

Sequence AL031723

Summer 2005

Kullback-Leibler Divergence

p ln (p/0.03) + (1-p) ln ((1-p)/(1-0.03))

p

KL

Div

erge

nce

Summer 2005


Kullback-Leibler: 0.508 Our Obs/Exp: 0.0029 C+G: 0.492

Summer 2005



Summer 2005


200 steps later…Kullback-Leibler: 0.520 Our Obs/Exp: 0.0033 C+G: 0.712

Summer 2005


600 steps later…Kullback-Leibler: 0.510 Our Obs/Exp: 0.0030 C+G: 0.598

Summer 2005

Our MethodK

L D

iver

gen c

e*1 2

/Obs

-Exp

rat

io*1

6 0/C

+G

Co n

tent

Base Position

Kullback-Leibler Divergence Observed/Expected Ratio C+G Content

Sequence AL031723

Summer 2005

Comparison of AL031723

Traditional

Method

Our Method

Summer 2005


Traditional

Method

Possible CpG Islands

3878-4534 5849-6136

6541-6820 8479-8698

10745-11049 18435-19580

25131-26359 35182-35441

36245-36576 36827-37606

Actual CpG Islands

18928-19547

25201-26371

36997-37693

Summer 2005


Our Method

Possible CpG Islands

19227-19435

25197-26147

36982-37420

Actual CpG Islands

18928-19547

25201-26371

36997-37693

Summer 2005

Cons

• Traditional Method

– Criteria not stringent enough

– If the expected part of the Obs/Exp ratio is unusually high then a high CpG count may not bring ratio above the cutoff

• Our Method

– Criteria sometimes too stringent

Summer 2005

Future Plans• CpG Islands

• Linkage Disequilibrium and SNPs

–Statistical analysis of the linkage disequilibrium coefficient

–Kullback-Leibler Divergence II

Summer 2005

Thank you!

• Former researchers– Andrew Dittmore

– Yasuhiro Goda

– Nick Heppenstall

– Michal Dvir

• Deborah Lycan• John S. Rogers Program

summer 2005 show me the cpg islands! alicia laughton (mathematics ‘06) jessica minnier...

Documents

end cpg islands

cpg islands genes gene

cpg islands regions

dna sequence slide

genetic information

statistical significance

high g c content

methylation causes c