Electronic supplementary material 1

Accurate CpG and non-CpG cytosine methylation analysis by

high-throughput locus-specific pyrosequencing in plants

Alexandre How-Kit1†

, Antoine Daunay1, Nicolas Mazaleyrat

2, Florence Busato

2,

Christian Daviaud2, Emeline Teyssier

3, Jean-François Deleuze

1,2, Philippe

Gallusci3 and Jörg Tost

2†

1 Laboratory for Functional Genomics, Fondation Jean Dausset - CEPH, 75010 Paris, France

2 Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-Institut de Génomique,

91000 Evry, France

3 UMR BFP, University Bordeaux 1, INRA, 78 Avenue E Bourleaux, 33883 Villenave d’Ornon, France

† Corresponding authors:

Alexandre How-Kit, Ph.D., Laboratory for Functional Genomics, Fondation Jean Dausset - CEPH,

Paris, F-75010, France, Tel.: +33-(0)1- 53725146, email: ahowkit@cephb.fr

Jörg Tost, Ph.D., Laboratory for Epigenetics and Environment, Centre National de Génotypage, CEA-

Institut de Génomique, Bâtiment G2, 2 rue Gaston Crémieux, CP 5721, 91057 Evry Cedex, France,

Tel.: +33-(0)1-60878423, Fax: +33-(0)1-60878485, email: tost@cng.fr

Supporting Information

NOR – promoter sequence

TAATTCAAAAGCAAATGAAGGACCATTCAAAAATTGTCCAAGTTAGGGCTACTAATTTTGAAATAGATTCCACTTGCTTTTTTCTTTAATTAGG

TAAGTGCAACATCCATAAATATTTTTCAAGATATTTTTCCGTAATTCATTCACAATTTTTTTTAAAAGAATTATTTAGTGTTTACCTGTTTACA

TTTATATTGAAATTAATTAAATTTAAAATCATAATTAAGAGTGGAGAAATTTCATTTATCAATAACCTAAATACTTAATTCTCCAATGAAACTA

ACTATAAGATTTTTCTCCCTAATAATAGGTTCATTTATTTTAGATTGGTCATGTGAAGGATATGTGTTGAAACAAATCCCTAAATTTCTTATTC

TTGTTAAGTTTAAAAATAAAAATGCTAAAAAAATTTTTTACAATGAAAGAATATTATAAAACTAATTAAATCCGTCCAAATTATAT<CATACGT

ATCGCGAGGATTCATCATAAATTGACTACTAGTCGTCGTATTTGTTGTCTCTATCCGAGTTCAAGATCAACG>ATACTATATATAAATACATCG

ATAGAGACAAGTTGTTGTAAAAAAATT[CCAAGTGTGACACTAGGCCAAATTTGTCCAAAGAGTAGCCTAGAAATGCTTTCTTATCTTATTATC

TGTCTGTCGAGTCATGTCAAACTTCTGTTGTAAAATTTAATCACTCCTAATTCGATATTTCAAAATCGAGTTTTGCAAATTAAGAAAATTTCAT

TGTAGGACGAGTTTCCTCTTTAATTAAATGATTCATATGAGCGCCACGAATTCAACTGAATATCAAATAAAATACTG]AATGATTGATATAGAT

CTCTTTATATATCTTGTGCAGGATAAAGTAGTTCTGCGTATATG<CCCCTTTTACTCGATTGTCCACGTGTTGGTACCAACTTGCATGCGTATC

G>ATTAATTATATTGCCTAATTTTCAGTTATCAAGCTCTAATTACATCATTGTCATGTATTAATCTAATCACCTCTTCAATTTATGCTAATGAC

GACCTCCACTTCTAATTTAATATTAACATATACACTATTTATTTTTCCACTAACGACTAATTTTTTTAATTTTTTTTTGACAATATTTATATAG

TAA

Figure S1: Input sequence for the design of amplification primers on bisulfite-treated DNA of the NOR promoter using PRIMER3. “[xxx]” denotes the region of interest, which must be included in the amplification product, while “<xxx>” indicate regions excluded for primer design. CpG dinucleotides are highlighted in gray and cytosines in other sequence contexts are highlighted in light gray. Framed nucleotides indicate the input polymorphisms used for the design of the pyrosequencing primer. Forward and reverse PCR primers are underlined.

NOR – promoter sequence (Solyc10g006880) -1939 from TSS TTGTTAAGTTTAAAAATAAAAATGCTAAAAAAATTTTTTACAATGAAAGAATATTATAAAACTAATTA

AATCCGTCCAAATTATATCATACGTATCGCGAGGATTCATCATAAATTGACTACTAGTCGTCGTATTT

GTTGTCTCTATCCGAGTTCAAGATCAACGATACTATATATAAATACATCGATAGAGACAAGTTGTTGT

AAAAAAATTCC1AAGTGTGAC2AC3TAGGCC4AAATTTGTCC5AAAGAGTAGCC6TAGAAATGCTTTC7T

TATC8TTATTATC9TGTC10TGTC11GAGTC12ATGTC13AAACTTC14TGTTGTAAAATTTAATC15ACTC

C16TAATTC17GATATTTC18AAAATC19GAGTTTTGC20AAATTAAGAAAATTTC21ATTGTAGGAC22GA

GTTTCCTC23TTTAATTAAATGATTCATATGAGCGCCACGAATTCAACTGAATATCAAATAAAATACTG

AATGATTGATATAGATCTCTTTATATATCTTGTGCAGGATAAAGTAGTTCTGCGTATATGCCCCTTTT

ACTCGATTGTCCACGTGTTGGTACCAACTTGCATGCGTATCGATTAATTATATTGCCTAATTTTCAGT

TATCAAGCTCTAATTACATCATTGTCATGTATTAATCTAATCACCTCTTCAATTTATGCTAATGACGA

CCTCCACTTCTAATTTAATATTAACATATACACTATTTATTTTTCCACTAACGACTAATTTTTTTAAT

TTTTTTTTGACAATATTTATATAGTAATTT

-1187 from TSS

CNR – promoter sequence (Solyc02g077920) -737 from TSS ATAGATTCACTGAACCATGCAATTCTATACCGTTCACTTCCAATAAATAAAACATAATACACTATGTT

TAGAGTACAAGACTCTCCTTGCTTGAAAAGGACTACCAAGTAGGGGTTACTGCAGTGACTACCAAGTA

GGGGTTACTGCAGTCATTTGTTAATTCATTTTGAGTAATGTAACTGTGCAAGATAATGATGTTTTTTT

TCCTTTTTGGTTAACTAAGATAATGATGGATTTAGGTAGATGAATTAGAC1ATC2TAGTGATAATGGAG

AGCC3AGTGC4AAC5AATTGAGC6ACTC7TAC8TGGACC9GAC10ATGGAC11AAC12TGAGAGACC13AAC

14TTGC15AGTATTATAATAGTGC16AAATTATAGTTTAGTC17GACTCCCTTC18GGAATC19TAC20TAC2

1ATAAAGAAC22TACC23ATAAAC24TATGTTAGATGGC25TATTAC26GGAGTTTAAATTAAACTC27GAA

AATATC28AGAAAAAGAAGTAACTTC29AACC30AATTAC31AATGC32ATACCC33TTATC34AC35AAGTG

AAAAAGAGTAAAC36GTGCC37AAACTC38TTTTGATCCCTCC39AAAGCTAGAGGAAAAGAGTGAGCAAT

TCACTACAAACCACTGGCTTTGGTCTATGGTTGACACAACTCCTCGGCTAATTGGTCAAAATATCTTG

TGACCACCAACCAGCAAGCACTAAATTGGATGTTCTATCAGCTTCTTTACATCATAAAACAGTGAATA

CTGAACGCTGAGAGGCTAACTGACTGCCCAAAAAAACCTTGACAAAAAGTTAGTGGAGTAACTACCTA

GGAGTAAATTCAATAGTAGACCTTGAAAAGAACTTTAGCAAAGTCATCATAAATGCTCTTCACGTCTC

ATGTACTAT

+111 from TSS

Figure S2: Sequence of the bisulfite PCR and pyrosequencing assays for the analysis of the methylation of all

cytosines in the NOR and CNR promoter. Each cytosine or cytosine group is highlighted in bold, yellow framed

and numbered. PCR primers are in bold blue (pre-bisulfite PCR primer) or red (bisulfite PCR primers) and

pyrosequencing primer are underlined.

Figure S3: Comparison of the quantification of the differences in cytosine methylation at CpG positions

between the developed Microsoft Excel VBA and the commercial PyroMark CpG Software analysis of the NOR

(A) and the CNR (B) promoter using the DNA methylation standards. As only CpG sites can be the analyzed by

the latter, only these common cytosine sites between the two methods corresponding to the “YG” sites of the

“Sequence to analyze” were compared by subtracting the mean cytosine methylation value estimated by the

VBA from the PyroMark CpG Software value.

-10

-5

0

5

10

C2 C11 C12 C13 C15 C17 C18 C19 C20 C21 C22

0%

25%

50%

75%

100%

-10

-5

0

5

10

C1 C4 C5 C6 C10 C11 C15 C16 C17 C21 C26 C28 C31 C32 C34 C35 C36

0%

25%

50%

75%

100%

Cyt

osi

ne

met

hyl

atio

n(%

)C

yto

sin

e m

eth

ylat

ion

(%)

A

B

Figure S4: Determination of the optimal annealing temperature for the amplification of the NOR (A) and CNR

(B) promoter on bisulfite-treated DNA using a fully methylated DNA sample (gDNA) and a fully unmethylated

DNA sample (bisulfite-converted WGA). The tested annealing temperatures are specified at the bottom of the

agarose gel electrophoresis.

gDNA

Bisulfite-conveted

WGA

49.8 50.3 51.5 53.4 55.7 58.3 61 63.7 66.1 68.0 69.4 °C

A B

gDNA

Bisulfite-conveted

WGA

49.8 50.3 51.5 53.4 55.7 58.3 61 63.7 66.1 68.0 69.4 °C

Figure S5: Annealing temperature-dependent quantification of the cytosine methylation level by bisulfite

pyrosequencing using a 50% DNA methylation standard and different annealing temperatures. Median value of

the DNA methylation level of the 20 first cytosines of the NOR (A) and the CNR (B) promoter. Quantification of

the DNA methylation degree of the 20 first cytosines in the NOR (C) and the CNR (D) promoter. The first

pyrosequencing primer of each assay was used to evaluate the methylation of the 20 first cytosines.

5453

5449

46 4749

63

0

10

20

30

40

50

60

70

80

90

100

49.8°C 50.3°C 51.5°C 53.4°C 55.7°C 58.3°C 61°C 63.7°C

7976

7467

6057 57

65

0

10

20

30

40

50

60

70

80

90

100

49.8°C 50.3°C 51.5°C 53.4°C 55.7°C 58.3°C 61°C 63.7°C

Cyt

osi

ne

met

hyl

atio

n(%

)

Cyt

osi

ne

met

hyl

atio

n(%

)

Annealing temperature Annealing temperature

30

40

50

60

70

80

49.8°C 50.3°C 51.5°C 53.4°C 55.7°C 58.3°C 61°C 63.7°C

CC1

C2

C3

CC4

CC5

CC6

CC7

C8

C9

C10

C11

C12

C13

CC1440

50

60

70

80

90

49.8°C 50.3°C 51.5°C 53.4°C 55.7°C 58.3°C 61°C 63.7°C

C1

C2

CC3

C4

C5

C6

CC7

C8

CC9

C10

C11

C12

CC13

C14

C15

C16

C D

Cyt

osi

ne

met

hyl

atio

n(%

)

Cyt

osi

ne

met

hyl

atio

n(%

)

Annealing Temperature Annealing Temperature

A B

rbcL – CDS sequence (LyesCp029) 56687 ATTATACTCCTGAGTACCAAACCAAGGATACTGATATATTAGCAGCATTCCGAGTAACTCCTCAACCT

GGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCTACTGGTACATGGACAACTGT

ATGGACCGATGGACTTACCAGTCTTGATCGTTACAAAGGGCGATGCTACCGCATCGAGCGCGTTGTTG

GAGAAAAAGATCAATATATTGCTTATGTAGCTTACCCTTTAGACCTTTTTGAAGAAGGTTCCGTTACC

AATATGTTTACTTCCATTGTAGGTAACGTATTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGA

TCTGCGAATCCCTCCTGCTTATGTTAAAACTTTCCAAGGTCCGCCTCATGGGATCCAAGTTGAAAGAG

ATAAATTGAACAAGTATGGTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCA

AAAAACTACGGTAGAGCTGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACCAAAGATGATGAGAA

CGTGAACTCACAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTTTAAAG

CACAGACTGAAACAGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGTACATGCGAAGAAATG

ATCAAAAGAGCTGTATTTGCTAGAGAATTGGGCGTTCCGATCGTAATGCATGACTACTTAACGGGGGG

ATTTACCGCAAATACTACCTTGGCTCATTATTGCCGAGATAATGGTCTACTTCTTCACATCCACCGTG

CAATGCATGCGGTTATTGATAGACAGAAGAATC1ATGGTATCC2ACTTCC3GGGTATTAGC14AAAAGC5

GTTAC6GTATGTC7TGGTGGAGATC8ATATTC9ACTC10TGGTACC11GTAGTAGGTAAAC12TTGAAGGT

GAAAGAGACATAACTTTGGGCTTTGTTGATTTACTGCGTGATGATTTTGTTGAACAAGATAGAAGTCG

CGGTATTTATTTCACTCAAGATTGGGTCTCTTTACCAGGTGTTCTACCTGTGGCTTCAGGAGGTATTC

ACGTTTGGCATATGCCTGCTCTGACCGAGATCTTTGGGGATGATTCCGTACTACAGTTCGGTGGAGGA

ACTTTAGGACATCCTTGGGGTAATGCGCCAGGTGCCGTAGCTAATCGAGTAGCTCTAGAAGCATGTGT

AAAAGCTCGTAATGAAGGACGTGATCTTGCTCGGGAAGGTAATGAGATTATTCGCGAGGCTTGCAAAT

GGAGCCCGGAACTAGCTGCTGCTTGTGAGGTATGGAAAGAGATCGTATTTAATTTTGCAGCAGTGGAC

GTTTTGGATAAGTAA

58120

Figure S6: Sequence of the bisulfite PCR and pyrosequencing assay for the analysis of the methylation of all

cytosines in the rbcL gene. Each cytosine or cytosine group is highlighted in bold, yellow framed and numbered.

PCR primers are in bold and in red (bisulfite PCR primers) and the pyrosequencing primer is underlined.

Figure S7: Comparison of cytosine methylation patterns of the NOR promoter in tomato pericarp during fruit development using publically available whole genome bisulfite sequencing data (Zhong et al. 2013) (A) and pyrosequencing results of the corresponding cytosines (B). For each cytosine analyzed by high-throughput locus-specific pyrosequencing, WGBS data was available in the Tomato Epigenome Database (http://ted.bti.cornell.edu/epigenome/index.html). Their localization (black horizontal line) is presented in the upper panel using the Tomato Epigenome Database genome browser including RIN ChIP-seq data for the identification of the RIN binding sites. When two or more cytosines could not be analyzed individually by pyrosequencing, their corresponding WGBS methylation percentages were averaged to allow comparison. Pyrosequencing data is the same as presented in Figure 6B without the 85 dpa stage. The dots under the cytosines indicate either a CpG (1 dot) or a CHG site (2 dots). dpa = days post anthesis.

0

10

20

30

40

50

60

70

80

90

100

CC

1

C2

C3

CC

4

CC

5

CC

6

CC

7

C8

C9

C1

0

C1

1

C1

2

C1

3

CC

14

C1

5

CC

C1

6

C1

7

C1

8

C1

9

C2

0

C2

1

C2

2

CC

C2

3

17 dpa

Mature Geen (39 dpa)

Breaker (42 dpa)

Red Ripe (52 dpa)

●● ● ●● ● ● ●●●

Cyt

osi

ne

met

hyl

atio

n(%

)A

B

Cyt

osi

ne

met

hyl

atio

n(%

)

●● ● ●● ● ● ●●●

0

10

20

30

40

50

60

70

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90

100

CC

1

C2

C3

CC

4

CC

5

CC

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CC

7

C8

C9

C1

0

C1

1

C1

2

C1

3

CC

14

C1

5

CC

C1

6

C1

7

C1

8

C1

9

C2

0

C2

1

C2

2

CC

C2

3

20 dpa

35 dpa

Breaker (39 dpa)

Red Ripe (55 dpa)

Figure S8: Comparison of cytosine methylation patterns of the CNR promoter in tomato pericarp during fruit development using publically available whole genome bisulfite sequencing data (Zhong et al. 2013) (A) and pyrosequencing results of the corresponding cytosines (B). For each cytosine analyzed by high-throughput locus-specific pyrosequencing, WGBS data was available in the Tomato Epigenome Database (http://ted.bti.cornell.edu/epigenome/index.html). Their localization (black horizontal line) is presented in the upper panel using the Tomato Epigenome Database genome browser including RIN ChIP-seq data for the identification of the RIN binding sites. When two or more cytosines could not be analyzed individually by pyrosequencing, their corresponding WGBS methylation percentages were averaged to allow comparison. Pyrosequencing data is the same as presented in Figure 6B without the 85 dpa stage. The dots under the cytosines indicate either a CpG (1 dot) or a CHG site (2 dots). dpa = days post anthesis.

0

10

20

30

40

50

60

70

80

90

100

C1

C2

CC

3

C4

C5

C6

CC

7

C8

CC

9

C1

0

C1

1

C1

2

CC

13

C1

4

C1

5

C1

6

C1

7

CC

CC

C1

8

C1

9

C2

0

C2

1

C2

2

CC

23

C2

4

C2

5

C2

6

CC

27

C2

8

CC

29

CC

30

C3

1

C3

2

CC

C3

3

C3

4

C3

5

C3

6

CC

37

CC

38

CC

CC

C3

9

17 dpa

Mature Geen (39 dpa)

Breaker (42 dpa)

Red Ripe (52 dpa)

●● ●●●●●

●● ●● ● ● ● ● ●

Cyt

osi

ne

met

hyl

atio

n(%

)

0

10

20

30

40

50

60

70

80

90

100

C1

C2

CC

3

C4

C5

C6

CC

7

C8

CC

9

C1

0

C1

1

C1

2

CC

13

C1

4

C1

5

C1

6

C1

7

CC

CC

C1

8

C1

9

C2

0

C2

1

C2

2

CC

23

C2

4

C2

5

C2

6

CC

27

C2

8

CC

29

CC

30

C3

1

C3

2

CC

C3

3

C3

4

C3

5

C3

6

CC

37

CC

38

CC

CC

C3

9

20 dpa

35 dpa

Breaker (39 dpa)

Red Ripe (55 dpa)

Cyt

osi

ne

met

hyl

atio

n(%

)A

B

●● ●●●●●

●● ●● ● ● ● ● ●

Table S1: PCR and pyrosequencing primers

Application

Primer Name

Sequence

PCR amplification

Sl_NOR_F ATYGATAGAGAYAAGTTGTTGTAAA

Sl_NOR_R Biotin-ACRCARAACTACTTTATCCTRCACA

Sl_CNR_F2 TGATGGATTTAGGTAGATGAATTAG

Sl_CNR_R2 Biotin-TTRCTCACTCTTTTCCTCTARCTT

Sl_CNR_preamp_F GGGGTTACTGCAGTCATTTG

Sl_CNR_preamp_R TGGGCAGTCAGTTAGCCTCT

Sl_NOR_preamp_F TCCGAGTTCAAGATCAACGA

Sl_NOR_preamp_R GGTCGTCATTAGCATAAATTGAA

Sl_RBCL_F1 GYATGYGGTTATTGATAGAYAGAA

Sl_RBCL_R1 Biotin-TCAACAAARCCCAAARTTATRTC

Pyrosequencing

Sl_NOR_pyro_F1 GAGAYAAGTTGTTGTAAAAA

Sl_NOR_pyro_F2 TYAAAYTTYTGTTGTAAAAT

Sl_CNR_pyro_F1 ATTTAGGTAGATGAATTAGA

Sl_CNR_pyro_F2 AGTGYAAATTATAGTTTAGT

Sl_CNR_pyro_F3 ATYAYAAGTGAAAAAGAGTA

Sl_RBCL_pyrod1d1 TATTGATAGAYAGAAGAAT

Table S2: Principle of the developed Microsoft Excel Visual Basic Applications for the design of the

pyrosequencing assay and quantification of the cytosine methylation level for all possible sequence contexts of

a cytosine site

Cytosine site

Genomic Sequence segment1

Sequence to Analyze

Dispensation Order2

Formula used for C methylation quantification from Dispensation Order peak intensities

Pyromark CpG Software

Quantification3

Standalone Cytosine

>CG >YG >TCG C/(C+T) Yes

>CA >YG*A >TCG*A C/(C+T) Yes

>T1

NCG >T1

NYG >TCG C/((C+T)/(1+(1N))) Yes

>T1

NCA >T1

NYG*A >TCG*A C/((C+T)/(1+(1N))) Yes

>CT1

NG >TT1

NG >TCTG C/((C+ΣT)/(1+(1N))) -

>CT1

-NA >TT1

NA >TCTA C/((C+ΣT)/(1+(1N))) -

>T1

NCT1-MG >T1

NTT1

MG >TCTG C/((C+ΣT)/(1+(1N)+(1M)) -

>T1

NCT1

MA >T1

NTT1

MA >TCTA C/((C+ΣT)/(1+(1N)+(1M)) -

ACG AYG ATCG C/(C+T) Yes

GCG GYG GTCG C/(C+T) Yes

ACA AYG*A ATCG*A C/(C+T) Yes

GCA GYG*A GTCG*A C/(C+T) Yes

AT1

NCG AT1

NYG ATCG C/((C+T)/(1+(1N))) Yes

AT1

NCA AT1

NYG*A ATCG*A C/((C+T)/(1+(1N))) Yes

GT1

NCG GT1

NYG GTCG C/((C+T)/(1+(1N))) Yes

GT1

NCA GT1

NYG*A GTCG*A C/((C+T)/(1+(1N))) Yes

ACT1

NG ATT1

NG ATCTG C/((C+ΣT)/(1+(1N))) -

ACT1

NA ATT1

NA ATCTA C/((C+ΣT)/(1+(1N))) -

GCT1

NG GTT1

NG GTCTG C/((C+ΣT)/(1+(1N))) -

GCT1

NA GTT1

NA GTCTA C/((C+ΣT)/(1+(1N))) -

AT1

NCT1

MG AT1

NTT1

MG ATCTG C/((C+ΣT)/(1+(1N)+(1M)) -

AT1

NCT1

MA AT1

NTT1

MA ATCTA C/((C+ΣT)/(1+(1N)+(1M)) -

GT1

NCT1

MG GT1

NTT1

MG GTCTG C/((C+ΣT)/(1+(1N)+(1M)) -

GT1

NCT1

MA GT1

NTT1

MA GTCTA C/((C+ΣT)/(1+(1N)+(1M)) -

Multiple Cytosines

>Y2

N4G >T2

NG >, succession of T and C5, then G (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

>Y2

N4A >T2

NA >, succession of T and C5, then A (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

AY2

N4G AT2

NG A, succession of T and C5, then G (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

AY2

N4A AT2

NA A, succession of T and C5, then A (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

GY2

N4G GT2

NG G, succession of T and C5, then G (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

GY2

N4A GT2

NA G, succession of T and C5, then A (ΣC/nb of initial C)/((ΣC+ΣT)/(2N)) -

“>” indicates the beginning of the pyrosequencing assay corresponding to the 3’ end of the pyrosequencing primer

“*” indicates a G present in the “Sequence to Analyze” and “Dispensation Order” but not in the original sequence

“0,1 or 2

N, M or P” indicates a number of repetitions ranging from 0, 1 or 2 to N, M or P 1 a genomic sequence segment is delimited by the beginning of the pyrosequencing assay and/or an “A” and/or a “G” nucleotide 2 when a nucleotide is repeated more than four times in the original sequence, a supernumerary nucleotide is added in the dispensation

sequence just following the same nucleotide i.e. ATTTTTTGGA ATTGA 3 some cytosine sites can also be quantified by PyroMark CpG Software 4 a Y in the original genomic sequence indicates a C or a T nucleotide where at least two C’s must be present to be considered as a multiple

cytosine site. For example, >YYYG can correspond to >TCCG, >CCTG, >CTCG or >CCCG in the original genomic sequence but not to >CTTG,

>TCTG or >TTCG 5 The succession of T and C depends on the original genomic sequence where the original T0

NCCT0

M sequences are converted into a TCT

dispensation order, original T0

NC1

2T1

MC and T0

NCCC are converted into a TCTC dispensation order, original T0

NC1

2T1

MC1

2T1

P,

T0

NCCCT1

M and T0

NCCCC are converted into a TCTCT dispensation order, and so on.

References

Zhong S et al. (2013) Single-base resolution methylomes of tomato fruit development reveal epigenome modifications associated with ripening Nat Biotechnol 31:154-159 doi:10.1038/nbt.2462