Electronic supplementary material 1 - Springer10.1007/s11103-015-0336... · Electronic...
Transcript of Electronic supplementary material 1 - Springer10.1007/s11103-015-0336... · Electronic...
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: [email protected]
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: [email protected]
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
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
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