em-seq wgbs dna weight 50ng 150ng 400ng 25ng...em-seq wgbs dna weight 25ng 50ng 150ng 400ng pcr...
TRANSCRIPT
EM-seq WGBS
DNA weight
50ng25ng 400ng150ng
PCR cycle
X 18X 12X 6
DNA weight
50ng25ng 400ng150ng
PCR cycle
X 18X 12X 6
Figure S1 Schematic diagram of experimental design.
0.0
2.5
5.0
7.5
ChrC
CG
(%)
0.0
2.5
5.0
7.5Ch
rC C
HG (%
)
0
2
4
6
8
ChrC
CHH
(%)
EM
WGBS
6.202e-14 6.202e-14 6.202e-14
Figure S2 CG, CHG, and CHH methylation levels detected in chloroplast genome by EM-seq and WGBS. Blue box represents EM-seq and red box represents WGBS. The P values, shown at the top of the plots, were estimated with a Student’s t-test.
0
1
CG
leve
l
0
0.7
CH
G le
vel
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0.14
CH
H le
vel
Chr. 1 Chr. 2 Chr.3 Chr. 4 Chr. 5
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1 Kb-1 Kb1 Kb-1 Kb1 Kb-1 Kb TE TE TE
CG
leve
l
CH
G le
vel
CH
H le
vel
0
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0.15
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1 Kb-1 Kb 1 Kb-1 Kb 1 Kb-1 KbGene body Gene body Gene body
CG
leve
l
CH
G le
vel
CH
H le
vel
a
b
c
400ng-12PCR-EM 400ng-12PCR-WGBS
Figure S3 Comparison of methylation pattern between EM-seq and WGBS libraries prepared with 400ng DNA input and 12 cycles of PCR. a Chromosomal distribution of CG, CHG, and CHH methyla-tions. Methylation levels were calculated with Arabidopsis chromosomes divided into 100 Kb bins. b Metaplots of CG, CHG, and CHH methylations over genes and 1 Kb flanking sequences. c Metaplots of CG, CHG, and CHH methylation over TEs and 1 Kb flanking sequences.
FWA
FWA
Chr. 4: 13,037,000 ~ 13,042,500CGCHGCHH
6X12
X18
X6X
12X
18X
WG
BS
EM-s
eq12
X12
XW
GB
SEM
-seq
0
150ng DNA
Chr. 4: 13,037,000 ~ 13,042,500CGCHGCHH
0
1400ng DNA
a
b
CGCHGCHHCGCHGCHH
CGCHGCHH
CGCHGCHHCGCHGCHHCGCHGCHH
Figure S4 Genome browser screenshots of FWA locus. a Comparison of methylation detected by EM-Seq and WGBS in libraries prepared with 50ng DNA input. b Comparison of methylation detected by EM-Seq and WGBS in libraries prepared with 400ng DNA input and 12 cycles of PCR.
229123
856
131 148
349
70 92
238138 105 120 87 126 119 116 99
285 276
159 156 145 116 85 72 70
299350 336 341
174 166
412 413
0
300
600
900 hypohyper
114 194
4965
113 139
2521
55 116
1134
112 85 106 55 78 68 38 33168 101 209 196 181 315 222 100 254 244 310 251 506 345 415
11651771
0
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6000
3115 33404418
2012 1931 23961785 1775 1992
2728 2306 2567 2541
1212 1071
3081 299134812503
6633
32543841
6937
19552863
7166
35864584 4432 4807
5729 5858
75568633
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CG
DM
R n
umbe
rC
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DM
R n
umbe
rC
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DM
R n
umbe
r
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b
c
hypohyper
CG
DM
R n
umbe
rC
HG
DM
R n
umbe
rC
HH
DM
R n
umbe
r
156 131 111 249 201 195 108 136 70 178 102 203 92 204 150 279 128186 221 271 234 290 346 310 355
1559
182 365 169646
219763
254
4183
0
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4500
256 434 562 1120 1029 2486338 531 185 394 137 1201 174 1035 207
2551147531 410 532 530 562 525 1396 1444
6916
377 1813 2263090
446
5601
486
31421
0
15000
35000
16623 2900546180 40292 47408
93284
18437 24771 12708 21324 698649073
783541285
7913
65724
3695
3234723580 20167 19406 19300
10967
57185 54049
138693
25276
99802
14413
143321
25077
194958
21592
441364
0
250000
500000
WGBS vs EM-seq hyper-CHG
Chr. 1
0
10
1
density
Chr. 2 Chr. 3 Chr. 4 Chr. 5
WGBS 400ng 12PCR CHG
EM-seq 400ng 12PCR CHG
Figure S5 DMR numbers and chromosomal distribution. a DMR numbers between EM-seq libraries prepared with different conditions. b DMR numbers between WGBS libraries prepared with different conditions. c Chromosomal distribution of CHG methylation in EM-seq and WBGS libraries prepared with 400ng DNA input and 12 cycles of PCR and WGBS hyper-CHG DMRs.
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EM WGBS
Chr
M C
G le
vel
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EM WGBS
Chr
M C
HG
leve
l
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EM WGBS
Chr
M C
HH
leve
l
b
35.23
35.41 35
.8735
.7535
.48 35.84
44.21
0
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chr1
chr2
chr3
chr4
chr5
chrC
chrM
GC
con
tent
(%)
a
Figure S6 Methylation in mitochondrial DNA. a GC contents of Arabidopsis chromosomes. b CG, CHG, and CHH methylation levels in mitochondrial DNA.
-5.0 center 5.0Kb
88.0
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89.0
25ng-6PCR-EM
-5.0 center 5.0Kb
PolV
pea
ks (n
=108
68)
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25ng-6PCR-WGBS
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25ng-12PCR-EM
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25ng-12PCR-WGBS
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25ng-18PCR-EM
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25ng-18PCR-WGBS
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-5.0 center 5.0Kb
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50ng-6PCR-EM
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50ng-6PCR-WGBS
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50ng-12PCR-EM
-5.0 center 5.0Kb
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50ng-12PCR-WGBS
-5.0 center 5.0Kb
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-5.0 center 5.0Kb
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8650ng-18PCR-EM
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-5.0 center 5.0Kb85
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50ng-18PCR-WGBS
-5.0 center 5.0Kb
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PolV
pea
ks (n
=108
68)
-5.0 center 5.0Kb
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150ng-6PCR-EM
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150ng-6PCR-WGBS
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150ng-12PCR-EM
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105150ng-18PCR-WGBS
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-5.0 center 5.0Kb88.5
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400ng-6PCR-EM
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400ng-6PCR-WGBS
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400ng-12PCR-EM
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400ng-18PCR-EM
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400ng-18PCR-WGBS
-5.0 center 5.0Kb
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Figure S7 Heatmaps showing read coverage of EM-seq (left six columns both rows) and WGBS (right six columns both rows) over PolV ChIP-seq peaks.
a
-5.0 center 5.0Kb86
88
90
400ng-12PCR-EM
-5.0 center 5.0Kbgene distance (bp)
Plot
ove
r EM
-Seq
hyp
er-C
G D
MR
(n=5
19)
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Cov
erag
e
Plot
ove
r EM
-Seq
hyp
er-C
G D
MR
(n=5
19)
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400ng-12PCR-WGBS
-5.0 center 5.0Kbgene distance (bp)
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erag
e
c
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erag
e
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Plot
ove
r EM
-Seq
hyp
er-C
HG
DM
R (n
=273
)
Cov
erag
e
-5.0 center 5.0Kb
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400ng-12PCR-EM
-5.0 center 5.0Kbgene distance (bp)
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Plot
ove
r EM
-Seq
hyp
er-C
HG
DM
R (n
=273
)
Cov
erag
ePl
ot o
ver E
M-S
eq h
yper
-CH
H D
MR
(n=8
23)
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400ng-12PCR-EM
-5.0 center 5.0Kbgene distance (bp)
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erag
ePl
ot o
ver E
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yper
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400ng-12PCR-WGBS
-5.0 center 5.0Kbgene distance (bp)
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d e
AT1G18880
Chr. 1: 6,520,700 ~ 6,523,400
WG
BSEM
-seq
01
CG DMR
WG
BSEM
-seq
01
CG DMR
AT1TE62440
Chr. 1: 18,828,500 ~ 18,833,500b
Figure S8 EM-seq hyper-DMRs. a,b Genome browser screenshots of EM-seq hyper-DMRs in pericen-tromeric TE (a) and in gene (b). c-e Heatmaps showing read coverage of EM-seq (left panels) and WGBS (right panels) over EM-seq hyper-CG (c), CHG (d), and CHH (e) DMRs.
AT1G08460 (HDA8)
[0 - 1.00]
DMR DMR
Chr.1: 2,672,000 ~ 2,675,000
Leaf
Flow
er
CG
CHG
CHH
a
flower hyper gene body CG gene (n=428)
leaf_1_2_7d
leaf_7_petiole_17d
leaf_7_proximal_half_17d
leaf_7_distal_half_17d
flowers_stage9_21d
flowers_stage10_11_21d
flowers_stage12_21d
flowers_stage12_carpels_21d
−2
−1
0
1
2
leaf hyper gene body CG gene (n=389)
leaf_1_2_7d
leaf_7_petiole_17d
leaf_7_proximal_half_17d
leaf_7_distal_half_17d
flowers_stage9_21d
flowers_stage10_11_21d
flowers_stage12_21d
flowers_stage12_carpels_21d
−2
−1
0
1
2
c
flower hyper gene body CG gene (n=428)
0
0.25
0.5
Met
hyla
tion
leve
l
gene body 1 kb-1 kb
leaf hyper gene body CG gene (n=389)
0
0.25
0.5leaf_CGleaf_CHGleaf_CHH
flower_CGflower_CHGflower_CHH
Met
hyla
tion
leve
lgene body 1 kb-1 kb
b
Figure S9 Gene body methylated genes. a Genome browser screenshots of flower hyper-CG DMRs in a gene body methylated gene. b Metaplots of CG, CHG, and CHH methylations in leaf and flower samples over genes containing hyper-CG DMRs and 1 Kb flanking sequences. c Clustering of expression patterns of gene body methylated genes containing hyper-CG DMRs.