supplementary materials for · 2020. 10. 9. · reliable tumor detection by whole-genome...

advances.sciencemag.org/cgi/content/full/6/42/eabb5427/DC1

Supplementary Materials for

Reliable tumor detection by whole-genome methylation sequencing of cell-free DNA

in cerebrospinal fluid of pediatric medulloblastoma

Jia Li, Sibo Zhao, Minjung Lee, Yue Yin, Jin Li, Yubin Zhou, Leomar Y. Ballester, Yoshua Esquenazi, Roderick H. Dashwood, Peter J. A. Davies, D. Williams Parsons, Xiao-Nan Li*, Yun Huang*, Deqiang Sun*

*Corresponding author. Email: [email protected] (X.-N.L.); [email protected] (Y.H.);

[email protected] (D.S.)

Published 16 October 2020, Sci. Adv. 6, eabb5427 (2020) DOI: 10.1126/sciadv.abb5427

The PDF file includes:

Figs. S1 to S7 Legends for tables S1 to S3

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/6/42/eabb5427/DC1)

Tables S1 to S3

0

0.1

0.20.3

0.40.5

0.6

0.70.80.9

mC

G/C

G

MB-SHH MB-WNTMB-G4MB-G3

C

Fig S1

D

mC

G/C

G

0

0.2

0.4

0.6

0.8

1.0

MB_CSFMB_tumorNontumorCSF

Cerebellum

Cerebellum1

mCG/CG

Fre

qu

en

cy

0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

66

e+

06

8e

+0

61

e+

07 Cerebellum2

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

66

e+

06

8e

+0

6

mCG/CG0.0 0.2 0.4 0.6 0.8 1.00

.0e

+0

04

.0e

+0

68

.0e

+0

61

.2e

+0

7

mCG/CG

Fre

quency

0.0 0.2 0.4 0.6 0.8 1.0

0e+

00

2e+

06

4e+

06

6e+

06

8e+

06

1e+

07

mCG/CG0.0 0.2 0.4 0.6 0.8 1.00

.0e

+0

04

.0e

+0

68

.0e

+0

61

.2e

+0

7

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e+

00

2e+

06

4e+

06

6e+

06

8e+

06

mCG/CG0.0 0.2 0.4 0.6 0.8 1.00

.0e

+0

04

.0e

+0

68

.0e

+0

61

.2e

+0

7

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e+

00

4e+

06

8e+

06

Nontumor.CSF1

mCG/CG

Fre

qu

en

cy

0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

6

Nontumor.CSF2

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

6

Nontumor.CSF3

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

66

e+

06

8e

+0

6

Nontumor.CSF4

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e

+0

02

e+

06

4e

+0

66

e+

06

8e

+0

6

CS

F

Patient 1 Patient 2 Patient 3

CS

F

0%

10%

20%

30%

40%

No additional Centrifuge

additional Centrifuge

Precipitation

Uniq

uely

mapped

r

atio

s

0%

20%

40%

60%

80%

100%

1 2 3 4 5 6 7 8 9 10

duplication <

Read p

erc

enta

ge

A BT

issu

e/T

um

or


mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0200000

600000

1000000

mCG/CG0.0 0.2 0.4 0.6 0.8 1.0

0e+

00

1e+

05

2e+

05

3e+

05

4e+

05

Patient 2_treatment Patient 2_beforeRecurrence

E

1.0

0.1

0.3

0.5

0.7

0.9

F

MB H3K27ac peaks center

4 Kb-4 Kb

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Nor

mal

ized

5hm

C le

vel

CerebellumMB_tumor1MB_tumor2MB_tumor3MB_CSF1MB_CSF2MB_CSF3

MB tumor tissueMB CSF

TSS TTS-2 kb 2 kb0

0.2

0.4

0.6

0.8

1.0

mC

G/C

G

MB tumor tissueMB CSF

0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ize

5hm

C le

vel 1.2

TSS TTS-2 kb 2 kb

5mC 5hmC

G

H

0.00

0.25

0.50

0.75

1.00

MB.

G3

MB.

G4

MB.

SHHI

NF

MB.

WNT

Patient 1

0.00

0.25

0.50

0.75

1.00

Patient 2

0.00

0.25

0.50

0.75

1.00

Patient 3

MB.

G3

MB.

G4

MB.

SHHI

NF

MB.

WNT

MB.

G3

MB.

G4

MB.

SHHI

NF

MB.

WNT

Pears

on c

orr

ela

tion

I

J

05

10

15

35 100 200 300 400 600 2000 10380bp

[FU

]Upper marker

CSF cfDNA

Mappin

g r

atio

s

Num

ber

of C

pG

s

Num

ber

of C

pG

s

0%

20%

40%

60%

80%

100%

Figure S1. Quality control for DNA methylation and hydroxymethylation libraries.

A. Representative size distribution of CSF DNA from MB patients determined by BioAnalyzer.

B. Number of read duplications versus read percentage for analysis using 200 µL CSF.

C. Ratios of uniquely mapped reads for three conditions using 200 µL CSF: no additional centrifuge,

additional centrifuge, and precipitation.

D. LiBis-mediated improvement in the uniquely mapped read ratios, number of recovered CpGs, and the

number of CpGs covered ≥3X.

E. Histograms of DNA methylation ratio distributions for all analyzed samples.

F. M-bias plots for MB CSF samples.

G. The mean DNA methylation levels of all samples analyzed in this study (left) and previously published

MB tumor WGBS datasets (right).

H. DNA methylation ratios (left) and normalized 5hmC (right) signals across the gene body.

I. Pearson correlation of DNA methylation levels between individual MB tumors analyzed in this study and

previously published WGBS data.

J. 5hmC distributions within common H3K27ac peaks (9) among four MB subtypes in cerebellum (purple),

MB tumor (solid line), and MB CSF ctDNA (dashed line).

C

D

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Pateint 1

Patie

nt 2

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient 1

Patie

nt 3

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient 2

Patie

nt 3

Patient 1

Patie

nt 2

Patient 1 Patient 2

Patie

nt 3

Pearson r = 0.48 Pearson r = 0.54 Pearson r = 0.53

CSF

mCG/CG

mC

G/C

G

5hmC(Log2)

5hm

C(L

og2)

0 2 4 6 8 10

02

46

810

12

0 2 4 6 8 10

02

46

810

12P

atie

nt 3

0 2 4 6 8 10 12

02

46

810

12


02

Cereb

ellum

1

Cereb

ellum

2

MB_tu

mor1

MB_tu

mor2

MB_tu

mor3

MB_C

SF1

MB_C

SF2

MB_C

SF3

DNA

met

hylat

ion ra

tio

0

1

Cereb

ellum

1

Cereb

ellum

2

MB_tu

mor2

MB_C

SF2

MB_tu

mor3

MB_C

SF3

MB_tu

mor1

MB_C

SF1

5hm

C

Gene Promoter regions (TSS upstream 2 kb & downstream 1 kb)

E

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient 1

Pat

ient

2

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient 1

Pat

ient

3

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient 2

Pat

ient

3

CSF - WGBS on CGIPearson r = 0.88 Pearson r = 0.93 Pearson r = 0.94

mCG/CG

mC

G/C

G

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient1

Pat

ient

4

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient2

Pat

ient

4

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Patient3

Pat

ient

4


A B

Fig S2

F

0.00

0.25

0.50

0.75

1.00

Pea

rson

Cor

rela

tion

MB_tu

mor1v

s

Nontu

mor_C

SF

MB_tu

mor2v

s

Nontu

mor_C

SF

MB_tu

mor3v

s

Nontu

mor_C

SF

C

Figure S2. Correlation analysis of DNA methylation and hydroxymethylation among samples

A. Pearson correlation analysis of the DNA methylation status of common CpG sites in patient #4 (WNT)

between CSF ctDNA samples and tumor DNA20.

B. Pearson correlation of the DNA methylation levels of CpGs between MB tumors and Nontumor CSFs.

C. Scatterplots showing the correlation of CSF WBGS data (top) or CSF CMS-IP-seq data (bottom) between

any two MB patients.

D. Heatmaps representing the DNA methylation (left) and hydroxymethylation (right) in gene promoter

regions;

E. Scatterplots showing the correlation of DNA methylation levels between CSF ctDNA (patient #4) and

MB WNT tumors20 within CGI regions.

F. Scatterplots showing the correlation of DNA methylation levels within the CGI regions in CSF samples

between any two MB patients.

0 2 4 6 8

02

46

81

0

2 4 6 8 10

02

46

81

01

2

0 2 4 6 8 10

02

46

81

01

2


0.0 0.2 0.4 0.6 0.8 1.0

0.00.20.40.60.81.0

CSF

0.0 0.2 0.4 0.6 0.8 1.0

0.00.20.40.60.81.0

Tumor0.0 0.2 0.4 0.6 0.8 1.0

0.00.20.40.60.81.0

Common DMRs


Common DHMRs


Tumor

CSF

mCG/CG

mC

G/C

G

5hmC(Log2)

5h

mC

(Lo

g2

)

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

Cerebellum

0.0 0.2 0.4 0.6 0.8 1.0

0.0

0.2

0.4

0.6

0.8

1.0

CG

IP

atie

nt 1

Patie

nt 2

Patie

nt 3

All

Cp

Gs

mCG/CG

mC

G/C

G

Cerebellum > MB tumor

Cerebellar Purkinje cell-granule cell precursor cell signalinginvolved in regulation of granule cell precursor cell proliferation

Regulation of cerebellar granule cell precursor proliferation

Radial pattern formation

Negative regulation of Toll signaling pathway

Release of cytoplasmic sequestered NF-kappaB

Detection of stimulus involved in sensory perception

0 100 200 300-log10(Binomial p value)

Positive regulation of Schwann cell chemotaxis

Regulation of Schwann cell migration

Positive regulation of synapse maturation

Positive regulation of glial cell migration

Regulation of synapse maturation

Negative regulation of calcium ion import

Positive regulation of dendrite morphogenesis

0 40 80 120-log10(Binomial p value)

Cerebellum < MB tumorDMRs Cerebellum < MB tumor

Cerebellar Purkinje cell-granule cell precursor cell signalinginvolved in regulation of granule cell precursor cell proliferation

Smoothened signaling pathway involved in regulation of cerebellar granule cell precursor cell proliferation

Regulation of cerebellar granule cell precursor proliferation

Smoothened signaling pathway involved in ventral spinal cord patterningSmoothened signaling pathway involved in ventral spinal cordinterneuron specification

Spinal cord ventral commissure morphogenesis

0 100 200 300 400-log10(Binomial p value)

Fc receptor signaling pathway

Autophagy

Regulation of intrinsic apoptotic signaling pathway

Cytoskeleton-dependent intracellular transport

Establishment of protein localization to membranes

Regulation of gene expression, epigenetic

-log10(Binomial p value)

0 20 40 60

Cerebellum > MB tumor

DHMRs

A

B

C D

Fig S3


Figure S3. The characterization of shared DMRs and DHMRs between MB tumor and CSF.

A. Scatterplots showing the correlation of global DNA methylation (top) and DNA methylation at CGIs

(bottom) between individual MB tumor samples and normal cerebellum. A total of 9,698,887 CpG sites

(patient #1), 3,050,750 CpG sites (patient #2), and 9,798,172 CpG sites (patient #3) were analyzed by

global DNA methylation analysis (top). A total of 6,660 CGIs, 1,384 CGIs, and 7,833 CGIs were analyzed

(bottom).

B. Scatterplots showing the correlation of DNA methylation and hydroxymethylation in shared DMRs (top)

and DHMRs (bottom) identified in Figure 2A, B between normal cerebellum and MB tumor and between

normal cerebellum and MB CSF.

C-D. GREAT analysis results for shared DMRs (A) and DHMRs (B) identified in Figure 2A, B. Left: Hyper-

DMRs or hypo-DMRs in MB tumor. Bottom: Hyper-DHMRs or hypo-DHMRs in tumor.

Characteristic Hyper DHMRs Characteristic Hypo DHMRs

Den

sity

DMCNonDMCCerebellumMB_tumorMB_CSF

0.0 0.2 0.4 0.6 0.8 1.0

01

23

4D

ensi

tyDMCNonDMCCerebellumMB_tumorMB_CSF

Characteristic Hyper DMRs Characteristic Hypo DMRs

A B

D

E

C

F

51.1%

20.2%

2.9%

6.8%

4.4% 6.8%

7.8%

0%

0%

56%

1.5%4.6%

2.9%

25.6%

5.5%

3.9%

0%

0%

m

CG

/CG

(Cer

ebel

lum

- C

SF)

mCG/CG(Cerebellum - Tumor)

mCG/CG(Cerebellum - Tumor)

m

CG

/CG

(Cer

ebel

lum

- C

SF)

mCG/CG mCG/CG

5hmC(Cerebellum - Tumor)

5hm

C(C

ereb

ellu

m -

CS

F)

5hm

C(C

ereb

ellu

m -

CS

F)

5hmC(Cerebellum - Tumor)

Figure S4. DNA methylation or hydroxymethylation dynamics in overlapping DMRs or DHMRs

A–B. DNA methylation differences within the shared hyper- (A) or hypo-DHMRs (B) identified in Figure 2B.

A total of 31,345 or 1,014 CpG sites within hyper- or hypo-DHMRs, respectively, were analyzed. The

dashed lines mark differences in DNA methylation of 20% between cerebellum and MB samples (x-axes

are MB tissue; y-axes are CSF).

C–D. The distributions of DNA methylation levels at CpGs displaying differences of 20% (DMC: solid lines) in DNA methylation within hyper- (C) or hypo-DHMRs (D). Green,

cerebellum; red, MB tumor tissue; orange, MB CSF.

E–F. DNA hydroxymethylation differences within the 17,898 shared hyper-DMRs (E) or 1,777 hypo-DMRs

(F) identified in Figure 2A. The dashed lines represent FPKM differences in 5hmc signal of 5 between

cerebellum and MB samples (x-axes, MB tissues; y-axes, MB CSF), whereas nearly all DHMRs in the

genome have a FPKM difference >5 (Figure S5).

Frequency

0 20 40 60 80 100

0200

400

600

800

1000

1200

Frequency

0 20 40 60 80 100

0500

1000

1500

Frequency

0 20 40 60 80 100

010

2030

4050

Frequency

0 20 40 60 80 100

010

2030

4050

6070

Hyper DHMRs Hypo DHMRs

5hmC (Cerebellum - MB_tumor)

5hmC (Cerebellum - MB_CSF) 5hmC (MB_CSF - Cerebellum)

5hmC (MB_CSF - Cerebellum)

Den

sity

All 5hmC peaks

5hmC(Cerebellum - MB_tumor/MB_CSF)

Cerebellum - MB_CSFCerebellum - MB_tumor

A

B

Fig S5

Figure S5. The hydroxymethylation differences in all DHMRs.

A. Density curves of the differences of 5hmC enrichment signal between cerebellum and MB samples

(solid line, CSF; dashed line, tumor tissue). The red box highlights the genomic regions displaying a FPKM

difference in 5hmC signal of

A

Fig S6

chr13:34,064,800 - 34,065,000

STARD13

Cerebellum1Cerebellum2

Non-tumorCSF1Non-tumorCSF2Non-tumorCSF3Non-tumorCSF4

MB_tumor1MB_tumor2MB_tumor3

MB-CSF1MB-CSF2MB-CSF3MB-SHH1MB-SHH2MB-SHH3MB-SHH4MB-SHH5MB-SHH6MB-WNT1MB-WNT2MB-WNT3MB-WNT4MB-WNT5MB-G4.1MB-G4.2MB-G4.3MB-G4.4MB-G4.5MB-G4.6MB-G4.7MB-G4.8MB-G4.9MB-G4.10MB-G4.11MB-G4.12MB-G3.1MB-G3.2MB-G3.3MB-G3.4MB-G3.5MB-G3.6MB-G3.7MB-G3.8MB-G3.9MB-G3.10MB-G3.11

This

stu

dyE

GA

S00

0010

0056

1

NCOR2chr12:125,033,400 - 125,033,600

C

SF vs

. G3

C

SF vs

. G4

C

SF vs

. SHH

C

SF vs

. WNT

0.8

0.4

0.0

Pear

son

Cor

rela

tion

Patient 4

CerebellumNontumor_CSF

MB_CSFMB_tumor

SHHWNT

G3

G4

B

C

D

E

78

35

Value

Color Key

Cou

nt

Cer

ebel

lum

1C

ereb

ellu

m2

MB

_tum

or1

MB

_tum

or2

MB

_tum

or3

MB

_CSF

1M

B_C

SF2

MB

_CSF

3

Non

Tum

or_C

SF1

Non

Tum

or_C

SF2

Non

Tum

or_C

SF3

Non

Tum

or_C

SF4

Figure S6. DNA methylation levels at MB signature CpGs in WGBS data

A. Functional genome elements analysis of MB CSF signature CpGs.

B. Heatmap representing the DNA methylation levels of MB signature CpGs among 2 normal cerebellums,

4 nontumor CSF samples, 3 MB tumors and 3 MB CSF samples.

C. t-SNE analysis of the DNA methylation levels of the common MB signature CpG sites among previously

published MB tumor samples (WGBS) with known subtype information (12), MB tumor/CSF samples (this

study) and nontumor CSF samples. Cerebellum, green; nontumor CSF, dark green; WNT, purple; SHH,

maroon; G3, dark blue; G4, light blue; MB tumors (this study), red square; MB CSF (this study), orange

square.

D. Two UCSC genome browser views showing the high DNA methylation levels at CpGs (black box) in all

MB tumor and CSF samples compared to the low methylation levels in all cerebellum and Nontumor CSF

samples.

E. Pearson correlation analysis of DNA methylation status at the CpGs shared between the MB-CSF

signature CpGs and the subtype-specific CpGs in CSF samples (patient #4), as identified between patient

#4 CSF and public WGBS data from pediatric MB patients.

51

28

64

37

146 6,452 9,228

MB-CSF (SHH) signature

CpGs (6,598)

MB-CSF (WNT) signature

CpGs (9,374)

781

63

45

2

163,615 603,043 100,234

Cerebellum vs.

MB.WNT.CSF (766,658)

Hyper DMCs

Cerebellum vs.

MB WNT tumors (263,849)

54,274 2,966,716 161,195

Hypo DMCs

Cerebellum vs.

MB.WNT.CSF (3,020,990)

Cerebellum vs.


20,385 49,363 7,942

Cerebellum vs.

MB.WNT.CSF (69,748)

Hyper DMRs

Cerebellum vs.


4,948 350,902 16,766

Hypo DMRs

Cerebellum vs.

MB.WNT.CSF (355,850)

Cerebellum vs.


Cer

ebellum

1

Cer

ebellum

2

MB_W

NTtu

mor

1

MB_C

SF4

MB_W

NTtu

mor

2

MB_W

NTtu

mor

3

MB_W

NTtu

mor

5

MB_W

NTtu

mor

4

Cere

bellum

1

Cere

bellum

2

MB

_W

NT

tum

or1

MB

_C

SF

4

MB

_W

NT

tum

or2

MB

_W

NT

tum

or3

MB

_W

NT

tum

or5

MB

_W

NT

tum

or4

MB

_S

HH

tum

or1

MB

_S

HH

tum

or2

MB

_S

HH

tum

or3

MB

_C

SF

1

MB

_C

SF

2

MB

_C

SF

3

C D

A B

E

Fig S7

0.00

0.25

0.50

0.75

1.00

Cer

ebel

lum

CSF

Tum

oral

pha

beta

gam

ma

alph

abe

taga

mm

aal

pha

beta

delta

gam

ma

alph

abe

ta

cg27490391

chr13:40152186-40152187

0.00

0.25

0.50

0.75

1.00

cg27579805

chr17:76101306-76101307

0.00

0.25

0.50

0.75

1.00

cg27638288

chr11:12071945-12071946

G3 G4 SHH WNT Cer

ebel

lum

CSF

Tum

oral

pha

beta

gam

ma

alph

abe

taga

mm

aal

pha

beta

delta

gam

ma

alph

abe

ta

G3 G4 SHH WNT Cer

ebel

lum

CSF

Tum

oral

pha

beta

gam

ma

alph

abe

taga

mm

aal

pha

beta

delta

gam

ma

alph

abe

ta

G3 G4 SHH WNT

DN

A m

eth

yla

tion r

atio

MB-CSF (WNT) signature CpGs

(n = 9,374)

Overlapped 146 MB-CSF signature CpGs

between SHH and WNT

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

+++++++++++++++++++++++

+++++++++++

+++++++ +

+++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++

++++++++++++++++++++++++++++

+

+ + +

p = 0.025

0.00

0.25

0.50

0.75

1.00

0 5 10 15 20 25Time (years)

Surv

ival pro

bability

mCG/CG low

mCG/CG high

7 probes on C9orf3 gene body

12,836 9,437 15,491

Cerebellum vs.

MB SHH tumors (22,273)

Hyper DMRs Cerebellum vs.


3,243 16,698 18,471

Hypo DMRs

Cerebellum vs.


Cerebellum vs.


EGAS00001000561

106,078 210,022 157,771

Cerebellum vs.


Hyper DMCs

Cerebellum vs.


31,070 237,955 184,399

Hypo DMCs

Cerebellum vs.


Cerebellum vs.


F

G H

58

41

73

62

AgeValue

0 0.6

025

00

Color Key

Cou

nt

35 d

ays

2 y

ears

old

5 y

ears

old

16 y

ears

old

25 y

ears

old

55 y

ears

old

Figure S7. Identification of MB-CSF (WNT) signature CpGs

A. Venn diagram representation of the overlapped DMRs/DMCs between normal cerebellum and MB SHH

tumors and between normal cerebellum and MB WNT tumors.

B. Venn diagram representation of the overlapped DMRs/DMCs between normal cerebellum and MB

WNT tumors and between normal cerebellum and MB WNT CSF sample.

C. Heatmap representing the DNA methylation levels of MB-CSF (WNT) signature CpGs among two normal

cerebellums, five MB WNT tumors and one MB WNT CSF sample.

D. Venn diagram representation of the overlapped CpGs between MB-CSF (SHH) and MB-CSF (WNT)

signature CpGs.

E. Heatmap representing the DNA methylation levels of the overlapped 146 CpGs in (C) among two normal

cerebellums, three MB SHH tumors, three MB SHH CSF samples, five MB WNT tumors and one MB WNT

CSF sample.

F. Kaplan-Meier survival curves of MB patients were separated according to median mean methylation

ratio of the 7 adjacent probes (cg09863917, cg03456213, cg13761321, cg14582550, cg14375632,

cg05621843, cg17406248) on C9orf3 gene body regions.

G. Boxplots showing the DNA methylation levels of the three CpGs used in the multivariate Cox model in

the array dataset (8).

H. The DNA methylation level within MB-CSF signature CpGs in normal brain tissues at different ages.

Table S1. WGBS and CMS-IP library quality control and sequencing data statistics.xlsx

Table S2. Clinical_information.xlsx

Table S3. MB-CSF.signature.CpGs and CpGs associated with OS.xlsx

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