Figure S1-S5 and Table S1-S2
Figure S1-A. Intensity plot of human 9K CGI array after hybridization with 2 ug sonicated
genomic DNA from MGC-803 cell line. Signal intensity of cy3 versus cy5 for all CGI probes is
shown on the array. After normalization, the signal from the two channels shows a high correlation
(Pearson Correlation Coefficient) of 0.9975.
1
Figure S1-B. Box plot of CGI arrays after hybridization. The three boxes represent the total
intensity of the CGI array after hybridization using the MMASS-v1 method in which an
enzyme-digested product from genomic DNA from the MGC-803 cell line was amplified at three
different annealing temperatures. The results show an increase in the total intensity with an
increasing annealing temperature from 65°C to 72°C.
2
3
Figure S2. Deviation from the theoretical ratio of each external control after hybridization
resulting from MeDIP with different antibody incubation times. (Top) The deviation of external
controls decreased with a primary incubation time increasing from 2 hrs to 12 hrs. This indicated
the binding efficiency of methylated external controls was improved if the primary antibody
incubation time was increased. (Middle) Secondary antibody incubation time optimized as 1, 2, 4,
and 6 hrs. The deviation of external controls decreased to minima with the secondary antibody
incubation time from 2 to 4 hrs. Although the binding efficiency of methylated external controls
was improved when the secondary antibody incubation time was increased, unspecific binding
also rose correspondingly. (Bottom) The deviations of external controls resulting from each of the
methods, including MeDIP with secondary antibody incubation for 2, 3, 4 hrs, MMASS-v2, and
DMH-v2, respectively.
4
Bisulfite Sequencing Verification
100.00%
90.00%
94.12%
88.00%
80.00%
75.00%
70.00%
60.00%
50.00%
Unique in MMASS-v2
Unique in DMH-v2
Common in MMASS-v2
and DMH-v2
Bilsufite Sequencing Verification
100.00%
100.00%
90.00%
85.71%
85.71%
Unique in MMASS-v2
Unique in MeDIP
80.00%
70.00%
60.00%
50.00%
Common in MMASS-v2
and MeDIP
Figure S3. Bisulfite sequencing verification results. (Top) The verified positive rates of bisufite
sequencing are 88.00% in MMASS-v2 uniqueness, 75.00% in DMH-v2 uniqueness, and 94.12%
in common, respectively. (Bottom) The verified positive rates of bisufite sequencing are 85.71%
in MMASS-v2 uniqueness, 85.71% in MeDIP uniqueness and 100% in common, respectively.
5
18
16
14
12
10
8
6
4
2
0
Input
24
74
6_
Co
nn
ec
Cl
t
on
e0
09
81
Cl
9
on
e0
12
63
Cl
4
on
e0
00
78
Cl
4
on
U
e0
HN
03
86
hs
5
cp
g
U
00
HN
04
46
hs
6
cp
g0
00
04
73
Cl
on
e0
01
71
3
4
U
HN
hs
cp
g
00
0
39
9
cl
o
ne
00
12
13
e0
Cl
on
Cl
on
e0
12
97
3
IP
Figure S4-A. Evaluation of the efficiency of methylated DNA enrichment through quantitative
PCR. All the selected clones were validated as hypermethylated in the MGC-803 cell line by
bisulfite sequencing. The results of quantitative PCR show that MeDIP enriched all the methylated
DNA fragments several fold relative to equal amounts of input DNA.
18
16
14
12
10
8
6
4
2
0
GES-1
00
24
hs
74
cp
g0
00
39
96
Cl
on
e0
09
81
Cl
9
on
e0
12
63
Cl
4
on
e0
00
78
Cl
4
on
U
e
00
H
N
38
hs
65
cp
g
00
U
H
04
N
46
hs
6
cp
g0
00
04
73
Cl
on
e0
01
71
3
U
H
N
cl
on
e
12
13
4
Cl
on
e0
Cl
on
e0
12
97
3
MGC-803
Figure S4-B. Validation of the methylation differences of several selected clones between Ges-1
and MGC-803 through quantitative PCR. These differentially methylated clones were identified in
MMASS-v2 and verified with bisulfite sequencing, but ranked low in MeDIP. The results show
that the methylation differences of Ges-1 compared to MGC-803 of these clones was significant
and consistent with bisulfite sequencing.
6
Bisulfite Sequencing Verification
100.00%
85.71%
88.89%
85.71%
80.00%
52.17%
60.00%
40.00%
20.00%
0.00%
MMASSv2 Unique
(B value>0)
MeDIP Unique
(B value>0)
MeDIP Unique
(-2<B value<0)
MeDIP Unique
(-4<B value<-2)
Figure S5. Bisulfite sequencing verification results with B value in MeDIP decreased. The entire
clones selected for bisulfite sequencing verification include: 27 candidates from MMASSv2
unique with B value more than 0, 16 candidates from MeDIP unique with B value more than 0, 18
candidates from MeDIP unique with B value between 0 and -2, and 23 candidates from MeDIP
unique with B value between -2 and -4. The verified positive rates of bisufite sequencing are
85.71% in MMASS-v2 uniqueness (B value >0), 85.71% in MeDIP uniqueness (B value >0),
88.89% in MeDIP uniqueness (-2 < B value < 0) and 52.17% in MeDIP uniqueness (-4 < B value
< -2), respectively. The ture positive rates were similar when B value in MeDIP decrease from 0 to
-2, but sharply descends as B value decrease to -4.
7
Table S1. The theoretical ratio of methylated fragments compared to unmethylated fragments in
each DNA external control from X and Y sets.
M/UN
P1
P6
P7
P9
P12
P13
P14
X
10:1
10:0
2:8
1:9
10:0
1:0
0:10
Y
2:8
10:0
10:0
10:0
1:9
1:0
0:10
Table S2. The ratio of each DNA external control from X and Y sets after hybridization from each
method.
Methods
Sets
P1
P6
P7
P9
P12
P13
P14
MeDIP(IP/Input)
X
40:11
4:1
4:5
2:5
4:1
4:1
NA(0:2.5)
Y
4:5
4:1
4:1
4:1
2:5
4:1
NA(0:2.5)
MMASS(M/UN)
X
Y
10:1
1:4
10:0
10:0
1:4
10:0
1:9
10:0
NA(10:0)
1:9
NA(1:0)
1:10
NA(0:10)
NA(0:10)
DMH(M/M)
Y/X
1:5
1:1
5:1
10:1
1:10
1:1
NA(0:0)
8