1. DNA methylation coverage in two
tissues of the Pacific Oyster
Claire Ellis
Bioinformatics Terminal Product
3/14/13

2. DNA methylation patterns in
Crassostrea gigas
Epigenetics describes DNA
modifications that change gene
expression without altering
nucleotide sequence.
DNA methylation in organisms
is extremely diverse, variable
among species, and can change
genome function under CH3
external influences.
DNA methylation
Source:
http://www.nist.gov/pml/div689/dna

3. Sequencing Approaches
Bisulfite sequencing was used
Bisulfite sequencing
to examine DNA methylation
Cm= methylated cytosine
in gonad tissue
C= unmethylated cytosine
MBD-Seq was used to 5’ ACmGTTCGCTTGAG 3’
examine DNA methylation in 3’ TGCmAAGCGAACTC 5’
gill tissue (Mackenzie)
Bisulfite Treatment
5’ ACmGTTUGUTTGAG 3’
3’ TGCmAAGUGAAUTU 5’

4. Approach
Bisulfite converted reads aligned to genome and %
methylation value per base calculated by processing
alignments
methylKit is an R package for DNA methylation
analysis and annotation from high-throughput
bisulfite sequencing

5. methylKit
Goal: obtain methylation coverage and
examine differential methylation between
gonad and gill tissues
Read annotation files and perform basic statistical
analyses for differentially methylated regions or
bases

7. Methylation Statistics
Histogram of % CpG methylation Histogram of % CpG methylation
test test
Gonad 94.7
Gill 16.8
40000
200000
12.5
30000
150000
9.8
Frequency
Frequency
8.1
20000
100000
7.1
6.4
5.4
4.5
4 4.2
10000
3.8
50000
2.4 2.6 2.7
1.8 1.7 1.6 2
1.4
4.4 1
0 0 0 0 0 0.3 0 0 0 0 0 0.3 0 0 0 0 0
0
0
20 40 60 80 100 0 20 40 60 80 100
% methylation per base % methylation per base

8. Coverage Statistics
Histogram of CpG coverage Histogram of CpG coverage
test test
91.6 Gonad 16.9 Gill
40000
200000
13.6
12.7
12.5
30000
150000
9.4
Frequency
Frequency 8.5
100000
20000
7.6
5.5
50000
10000
3.9
2.9
2
7.2 1.5
1
0.70.5
0 0 0.7 0 0.2 0 0 0 0 0 0 0.1 0 0 0 0 0 0 0 0.30.20.10.1 0 0 0
0 0
0
0
0.0 0.5 1.0 1.5 2.0 1.0 1.5 2.0 2.5 3.0
log10 of read coverage per base log10 of read coverage per base

9. CpG base correlation
CpG base pearson cor.
0.0 0.2 0.4 0.6 0.8 1.0
1.0
~/Desktop/TJGR_GonadPE_BS_v9_90_CG_methylkit_modified.txt
0.8
0.6
Gonad 0.068 0.068
0.4
0.2
1.0
~/Desktop/TJGR_gillMBD_BS_v9_10x_methylkit_modified.tabular.txt
0.8
Gill
0.6
y
0.4
0.2
0.0
0.2 0.4 0.6 0.8 1.0

10. Methylation Clustering
CpG methylation clustering
0.8
0.6
Height
Blue= Gonad
0.4
Red= Gill
0.2
0.0
ethylkit_modified.txt
t_modified.tabular.txt
Samples
Distance method: "correlation"; Clustering method: "ward"

11. PCA- Principal Component Analysis
CpG methylation PCA Analysis
2e-12
~/Desktop/TJGR_GonadPE_BS_v9_90_CG_methylkit_modified.txt
1e-12
Blue= Gonad
0e+00
PC2
Red= Gill
-1e-12
-2e-12
-60 -40 -20 0 20 40 60
PC1

12. Conclusions
Additional analyses included examining type of differential
methylation (hypo and hyper)
Extracted bases with a q-value <0.01 and % methylation
difference >25%
The methylKit package was successfully used to
characterize DNA methylation
Differences between gonad and gill methylation profiles
may be due to library prep
Will use R script for future analyses comparing different
samples’ methylation profiles

13. Methylation Statistics
> getMethylationStats(gonad,plot=F,both.strands=F)
methylation statistics per base
summary (gonad):
Min. 1st Qu. Median Mean 3rd Qu. Max.
9.091 100.000 100.000 97.360 100.000 100.000
Percentiles (gonad):
0% 10% 20% 30% 40% 50% 60% 70% 80% 95% 99.5% 99.9% 100%
9.09 100 100 100 100 100 100 100 100 100 100 100 100
summary (gill):
Min. 1st Qu. Median Mean 3rd Qu. Max.
0.00 54.55 78.70 69.56 91.89 100.000
Percentiles (gill):
0% 10% 20% 30% 40% 50% 60% 70% 80% 95% 99.5% 99.9% 100%
0 21.4 46.6 61.1 70.9 78.7 84.6 90 93.7 100 100 100 100