SUPPLEMENTARY MATERIAL 1
ASSAY DESIGN REPORT
EpigenDx CpG Assay Design - Premium
info@epigendx.com
Assays/Assignment#
Page:
1(12)
Human HSD17B10/ADS2501 and ADS2502
Assay report:
Date:
Matthew Poulin
01-23-2012
1.
INTENT OF USE ............................................................................................................................................................ 2
2.
SUMMARY ...................................................................................................................................................................... 2
3.
HUMAN HSD17B10 CPG METHYLATION ASSAYS .............................................................................................. 3
3.1.
MATERIALS PROVIDED: ........................................................................................................................................... 3
3.2.
MATERIALS REQUIRED BUT NOT INCLUDED.............................................................................................................. 3
3.3.
HUMAN HSD17B10 PROMOTER AND SURROUNDING GENOMIC DNA SEQUENCE ................................................... 3
3.4.
BISULFITE CONVERTED TARGET SEQUENCES: ........................................................................................................... 4
3.4.1. ADS2502 ............................................................................................................................................................. 4
3.4.2. ADS2501 ............................................................................................................................................................. 4
3.5.
TARGET CPG SITES COORDINATES .......................................................................................................................... 5
3.6.
ASSAY TARGET SEQUENCES .................................................................................................................................... 5
4.
ASSAY VALIDATION ................................................................................................................................................... 6
4.1.
SELECTED ASSAY VALIDATION RESULTS ................................................................................................................ 6
4.1.1
ADS2502FS1 Validated by Pyrosequencing ....................................................................................................... 6
4.1.2
ADS2502FS2 Validated by Pyrosequencing ....................................................................................................... 7
4.1.3
ADS2501FS1 Validated by Pyrosequencing ....................................................................................................... 8
4.2.
PCR BIAS TESTING USING PYROSEQUENCING ......................................................................................................... 9
4.3.
EXAMPLE OF QUANTITATIVE ANALYSIS (IN % METHYLATION) ............................................................................... 9
5.
PROTOCOLS ................................................................................................................................................................ 10
5.1.
ASSAY TEMPLATE .................................................................................................................................................. 10
5.1.1
Template ............................................................................................................................................................ 10
5.1.2
Bisulfite modification ........................................................................................................................................ 10
5.2
PCR PROTOCOL ..................................................................................................................................................... 10
5.2.1
Recommended PCR protocol............................................................................................................................. 10
5.2.2
Recommended PCR cycling conditions: ............................................................................................................ 10
6.
PYROSEQUENCINGTM ANALYSIS .......................................................................................................................... 11
6.1.
PYROSEQUENCING PROTOCOL: .............................................................................................................................. 11
Explanations and abbreviations:
Tail: A short tail (eg. TTT) may have been added to the 5’-end of the non-biotinylated PCR primer in order to
reduce the possibility of self-priming in the PCR product.
Abbreviations: F=forward, R=reverse, P=PCR primer, S=sequencing primer, B=biotin.
For research use only. Not intended for diagnostic purposes.
These mutations may be covered by a patent or patent applications. This protocol is made available for scientific research
only and in no way confers the rights to perform this assay for commercial purposes or profit.
© Copyright 2006 EpigenDx.
Pyrosequencing, PSQ, Pyrogram and
are trademarks owned by Qiagen. Pyrosequencing technology
is covered by patents and patent applications owned by Qiagen.
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
1.
Intent of Use
HSD17B10 encodes 3-hydroxyacyl-CoA dehydrogenase type II, a
member of the short-chain dehydrogenase/reductase superfamily.
The gene product is a mitochondrial protein that catalyzes the
oxidation of a wide variety of fatty acids, alcohols, and steroids. The
protein has been implicated in the development of Alzheimer's
disease, and mutations in the gene are the cause of 2-methyl-3hydroxybutyryl-CoA dehydrogenase deficiency (MHBD). Several
alternatively spliced transcript variants have been identified, but the
full-length nature of only two transcript variants has been
determined. [provided by RefSeq, Jul 2008]. This gene is located at
Chromosome X: 53,458,206-53,461,320 reverse strand. There are 5
transcriptional variants of this gene.
This assay was designed for the analysis of DNA methylation. It is
for research use only and is NOT to be used in diagnostic procedures.
EpigenDx takes no responsibility for the use of these reagents in any
other context than specified in this manual.
2.
Summary
Assay ID
Gene Name
CpG Loci
Human hydroxysteroid
(17-beta)
ADS2502 dehydrogenase 10
Ensembl Gene ID:
ENSG00000072506
Human HSD17B10
Ensembl Transcript ID:
ENST00000168216
-171 to -7 from ATG
-143 to +22 from TSS
Human hydroxysteroid
(17-beta)
ADS2501 dehydrogenase 10
Ensembl Gene ID:
ENSG00000072506
Human HSD17B10
Ensembl Transcript ID:
ENST00000168216
+2259 from ATG
+2287 from TSS
Also Known
Type of
As
assay
ABAD; CAMR;
ERAB; HCD2;
Bisulfite
MHBD;
sequencing
HADH2;
MRPP2;
MRX17;
MRX31;
SCHAD;
Bisulfite
MRXS10;
sequencing
SDR5C1; 17bHSD10;
DUPXp11.22
PCR Size # of CpGs
217 bp
22
240 bp
1
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
3.
Human HSD17B10 CpG Methylation
Assays
3.1. Materials Provided:
Assay ID
CpG Loci
ADS2502FS1
Human HSD17B10
Ensembl Transcript ID:
ENST00000168216
-171 to -7 from ATG
-143 to +22 from TSS
CpG #1 to #3
ADS2502FS2
ADS2502
ADS2501
ADS2501FS1
Type of primer
Primer name
PCR F
ADS2502FP
PCR R
ADS2502RPB
Seq F
ADS2502FS1
CpG #4 to #23
Seq F
ADS2502FS2
Human HSD17B10
Ensembl Transcript ID:
ENST00000168216
+2259 from ATG
+2287 from TSS
CpG #1
PCR F
5' mod
biotin
ADS2501FP
ADS2501RPB
PCR R
Seq F
Biotin
ADS2501FS1
3.2. Materials required but not included
-
-
DNA extraction reagents.
PCR reagents
Pyrosequencing or downstream analysis reagents and
consumables.
3.3. Human HSD17B10 Promoter and Surrounding
Genomic DNA Sequence
ctgtactgtaattctctttctccacctggactgtgccctcctggaaggcatgaatcttttattattccg
ctgggtatcccaggtgccccgcacaccgtctagcatataggagtctcaaatacattcatacttgaatta
agactttttcagaggagaaaggcatccgttctcaatatccacccctcttcccccacgcggagcaggcca
cttgccaaaaggccagctagagagtagggcgaggggtgttgtggtattgtacttctgctattggtctct
ttccccagttgaaataactctggggataccttagcaggcggaatccgccctctggccaaaggactagcg
taccaggccacgcccccacgtctcatgcggcagcggcagacgccccggcccgtcgcatccgccccttcc
gccgcttcgcctcggccaatcaacgagcgcccgcgcccccatcCCCATCCCGTGGAGTGGCCGGCGACA
AGATGGCAGCAGCGTGTCGGAGCGTGAAGgtagaaggttctttgcctctgttgcttgccttcctttacg
ggccgggacagcgcggccgtggaactaggagaaagagatcggtttcgagggaaagggtcatgcttatgt
aggcctggtattgtggccgttgggctcaagaggtgaaagtcagcactccgcggcgcatctgtctgtctg
tctgtctgtctatctatctatctatcaatcatctatctatctatcgtcacgggacgggctgtctgggga
tgagatcccttctgcccccgaggtcccaacgctgaggagaagcagcacacctagtttacccgcaggggt
gaccacgacctctcccctccccccttctcctcagaagcctggagaacaggcaggcgacagcgtggggat
ggtctgggcaatgtaacagctgaccctgaccatgtcccttttgacagGGCCTGGTGGCGGTAATAACCG
GAGGAGCCTCGGGCCTGGGCCTGGCCACGGCGGAGCGACTTGTGGGGCAGGGAGCCTCTGCTGTGCTTC
TGGACCTGCCCAACTCGGGTGGGGAGGCCCAAGCCAAGAAGTTAGGAAACAACTGCGTTTTCGCCCCAG
CCGACgtaagtggggtcacctctcctctcccagggtgtgcatgaggtcaccaacaccggcctgggaacc
tgtgggggtccctcccataagggtctcctctatgggggcggtagacccatctccttgtctcttttggcc
tgagccttcaagcactgtgggaccacctgtgataaggattctctaaagctgggattcacaacaatgagg
atggggattggacgtaacaatctcctggaggagttatgtatggtttgaaaatgcattttgtgccttgta
attttatatttacaaagtcaaaaaacttgttttcatgaaacaaactacagaaagtgaaacttgatgaaa
ASSAY DESIGN REPORT
Assays/Assignment#
Human HSD17B10/ADS2501 and ADS2502
Page:
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tcatcttagcaggtaaggatacaaaaaagctagtttatcttcctatcaataattctcacaagagggtta
ttttttatttttatttttgagatggagtctcgctctgttgcccaggctggagtgcagtggcacaatctc
agctccttgcaacctccatcttctgggttcaagcgattttcctgcctcaacctcccaagtagctgggat
tacaggcacctgccaccacgcccagctaatttttttttttgtatttttagaagagatggggtttcacca
tgttggccgggctggtcttgaactcctgatctcacgtgatccgcccgcctcggcctcccaaagtgctgg
gattacaggtgtgagccactgcacccagcctcacaacagggttgcattagtgctctgatccacagcaat
taagaatcttcaggagcatgtgaatttatcaagaaggtattgatttgaaaaatgttttgcaacactgct
ctatattacctaatattttcacctttgacctactctctcaatttttttttttgggacagagtctcactg
ttgcccaggctgcagtgcaatggcacgatctcagctcactgcaacctctgcctcccgggttcaagcgat
tcttctgcctcagccttccaagtagctgggattacaggcatgtgtcactatgccggctaattttgtatt
tttagtagagacgggttttcaccacgttggccaggctggtctcaaactcctgatcttaagtgatccacc
agcctcggcctcccaaagtgctgagattacaggcatgagccaccgcacccagcctatctctcaagtttt
acaacctttctcttggcctctcccttctcacaaatctcttctcacccaaggactgggcatgccctgccc
tgccctccctcaggtttagtgggaagagtgggtgctgggagatgaataccttctccacttctctctttg
aagGTGACCTCTGAGAAGGATGTGCAAACAGCTCTGGCTCTAGCAAAAGGAAAGTTTGGCCGTGTGGAT
GTAGCTGTCAACTGTGCAGGCATCGCGGTGGCTAGCAAGACGTACAACTTAAAGAAGGGCCAGACCCAT
ACCTTGGAAGACTTCCAGCGAGTTCTTGATgtaaggccttggaagttctccagggatagtggtagtaag
aagtatctggctgtggaggaccccaaagtttttaggggacaaaggcttcttctgcctcaggtcctatag
catgtctccatctgtcctacagGTGAATCTCATGGGCACCTTCAATGTGATCCGCCTGGTGGCTGGTGA
GATGGGCCAGAATGAACCAGACCAGGGAGGCCAACGTGGGGTCATCATCAACACTGCCAGTGTGGCTGC
CTTCGAGGGTCAGgtgtgtgggcaggggtaagacttatgcctcctaagtgacttgttggggccctccca
cctatgacttctactctttctccagGTTGGACAAGCTGCATACTCTGCTTCCAAGGGGGGAATAGTGGG
CATGACACTGCCCATTGCTCGGGATCTGGCTCCCATAGGTATCCGGGTGATGACCATTGCCCCAGgtag
acatatcccctctctccatcatacctgggattgggtgggatccatggacagttgagaggggaaggtatc
caccacctaagcagcagcagccttttatctttgggccccagagaagcaccaa
-
-
Lower cases letters in green are 5’ upstream sequence.
Lower case letters in blue are the intron sequence.
Capital letters in purple are the exon 1 (5’UTR) sequence.
Capital letters in black are the exon 1 coding region
CpG sites in this sequence are red (CGs).
Green highlighted (C) is the transcriptional start site.
Green highlighted (ATG) is the translational start site (ATG).
ADS2501 target CpG loci are blue highlighted (CG).
ADS2502 target CpG loci are gray highlighted (CG).
3.4. Bisulfite converted target sequences:
3.4.1. ADS2502
taggyggaattygttttttggttaaaggattagygtattaggttaygtttttaygttttatgyggtagy
ggtagaygtttyggttygtygtattygtttttttygtygtttygtttyggttaattaaygagygttygy
gtttttattTTTATTTYGTGGAGTGGTYGGYGATAAGATGGTAGTAGYGTGT
3.4.2. ADS2501
TTGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGATTAGGGAGGTTAAYGTGGGGTTATTATTAA
TATTGTTAGTGTGGTTGTTTTYGAGGGTTAGgtgtgtgggtaggggtaa
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
3.5. Target CpG Sites Coordinates
From ATG
From TSS
GCRh36/hg18
X Chr
GCRh37/hg19
X Chr
ADS2502
-171 to -7
-143 to +22
ADS2501
+2259
+2287
53478188 to 53478024
53459035
53461463 to 53461299
53475759
3.6. Assay Target Sequences
Assay ID
ADS2502FS1
ADS2502FS2
Genomic Target Sequence
Bisulfite Converted Target
Sequence
caggcggaatccgccctctggccaa taggyggaattygttttttggttaa
aggactagcgtaccagg
aggattagygtattagg
ccacgcccccacgtctcatgcggca
gcggcagacgccccggcccgtcgca
tccgccccttccgccgcttcgcctc
ggccaatcaacgagcgcccgcgccc
ccatcCCCATCCCGTGGAGTGGCCG
GCGACAAGATGG (c>g,
rs1264014)
ADS2501FS1 TCCGCCTGGTGGCTGGTGAGATGGG
CCAGAATGAACCAGA (C>T,
rs28935475 lost CpG status)
ttaygtttttaygttttatgyggta
gyggtagaygtttyggttygtygta
ttygtttttttygtygt/gttygtt
tyggttaattaaygagygttygygt
ttttattTTTATTTYGTGGAGTGGT
YGGYGATAAGATGG
Pyrosequencing
Dispensation order
ATCATGTCGTATCGTC
TGCTAGACTATGTCGT
A
TCGATCGTCTGATCGT
CTATAGTCGTCATGTC
GTAGTATCAGTTCGTC
AGTCGTCGATCGTTCA
GTCGTTCTGTTCGTCA
TCGATCGATGTCAGTC
GTCGTCTATTGATTCG
TGAGTAGTCGTCGA
TYGTTTGGTGGTTGGTGAGATGGGT
ATCGCTGTGCTG
TAGAATGAATTAGA
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
4.
Assay Validation
4.1. Selected Assay Validation Results
4.1.1
ADS2502FS1 Validated by Pyrosequencing
Sequence to analyze:
TAGGYGGAATTYGTTTTTTGGTTAAAGGATTAGYGTATTAGG
(Yellow highlights are the controls for bisulfite modification completion)
Histogram:
6
5
4
3
2
1
0
A
T
C
A
T
G
T
C
G
T
5
A
T
C
G
T
10
Pyrogram:
Control: Low methylated DNA
B8 : TAGGYGGAATTYGTTTTTTGGTTAAAGGATTAGYGTATTAGG
1%
C
T
G
C
T
15
A
G
A
C
T
20
A
T
G
T
25
1%
5%
1500
1000
500
0
E
S
A
T
C
A
T
G
T
C
G
T
5
A
T
C
G
T
10
Control: Medium methylated DNA
E8 : TAGGYGGAATTYGTTTTTTGGTTAAAGGATTAGYGTATTAGG
34%
C
T
G
C
T
15
A
G
A
C
T
20
A
T
G
T
C
25
G
T
A
30
38%
35%
1200
1000
800
600
400
200
0
-200
E
S
A
T
C
A
T
G
T
C
G
5
T
A
T
C
G
T
10
Control: High methylated DNA
G8 : TAGGYGGAATTYGTTTTTTGGTTAAAGGATTAGYGTATTAGG
92%
C
T
G
C
T
15
A
G
A
C
T
20
A
T
G
T
C
25
G
T
A
30
91%
96%
1000
800
600
400
200
0
-200
E
S
A
T
C
A
T
G
T
C
G
T
5
A
T
C
G
T
10
C
T
G
C
15
Control: No template control
H8 : TAGGYGGAATTYGTTTTTTGGTTAAAGGATTAGYGTATTAGG
N/A
T
A
G
A
C
20
T
A
T
G
T
25
C
G
T
A
C
G
T
30
N/A
N/A
20
10
0
-10
-20
-30
E
S
A
T
C
A
T
5
G
T
C
G
T
10
A
T
C
G
T
15
C
T
G
C
T
20
A
G
A
C
T
25
A
T
G
C
30
T
30
A
G
T
A
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
4.1.2
ADS2502FS2 Validated by Pyrosequencing
Sequence to analyze:
TTAYGTTTTTAYGTTTTATGYGGTAGYGGTAGAYGTTTYGGTTYGTYGTATTYGTTTTTTTYGTYGT/GTTYG
TTTYGGTTAATTAAYGAGYGTTYGYGTTTTTATTTTTATTTYGTGGAGTGGTYGGYGATAAGATGG
(Yellow highlights are the controls for bisulfite modification completion)
Histogram:
8
6
4
2
0
T C G A T C G TC T G A TC G T C T A T A G T C G T C A T G T C G TA G T A T C A G T T C G T C A G TC G T CG A T CG T T CA G T C G T T C TG T T CG T C A T C G A TC G A TG T C A G T C G T C G TC T A T TG A T TC G T GA G T A G T C G TC G A
5
10
15
20
25
30
35
40
45
50
Pyrogram:
Control: Low methylated DNA
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
B10 : T T AYGT T T T T AYGT T T T AT GYGGT AGYGGTAGAYGTTTYGGTTYGTYGTATTYGTTTTTTTYGTYGT/ GTTYGTTTYGGTTAATTAAYGAGYGTTYGYGTTTTTATTTTTATTTYGTGGAGTGGTYGGYGATAAGATGG
3%
1%
1%
0%
3%
6%
2%
6%
2%
3%
2% N/A
2%
2%
2%
5%
0%
4%
4%
2%
1200
1000
800
600
400
200
0
-200
E S TCGA T CG TC T GA TCG T C TA TA G TCG TC A TG TCG TA G TA T CA G T T CG TCA G TCG TC G A TCG T T CA G TC G T TC TG T TCG TC A TCGA T CGA TG T CA G TC G TCG TC TA T TGA T TCG TG A G TA G TCG TCG A
5
10
15
20
25
30
35
40
45
50
Control: Medium methylated DNA
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
E10 : T T AYGT T T T T AYGT T T T AT GYGGT AGYGGTAGAYGTTTYGGTTYGTYGTATTYGTTTTTTTYGTYGT/ GTTYGTTTYGGTTAATTAAYGAGYGTTYGYGTTTTTATTTTTATTTYGTGGAGTGGTYGGYGATAAGATGG
40%
36%
33%
38%
41%
36%
36%
39%
38% 33%
30%N/A
37%
26%
29%
36% 26%
42%
36% 29%
1000
800
600
400
200
0
-200
E S TC GA TCG TC TGA TCG TC TA TA G T CG TCA TG TCG T A G TA T CA G T T CG TCA G TCG TC GA TCG T TCA G T CG T TC T G T TCG TCA TC GA TCGA TG TCA G TC G TC G TC TA T TGA T TCG TGA G TA G T CG TCGA
5
10
15
20
25
30
35
40
45
Control: High methylated DNA
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
G10 : T T AYGT T T T T AYGT T T T AT GYGGT AGYGGTAGAYGTTTYGGTTYGTYGTATTYGTTTTTTTYGTYGT/ GTTYGTTTYGGTTAATTAAYGAGYGTTYGYGTTTTTATTTTTATTTYGTGGAGTGGTYGGYGATAAGATGG
95%
86%
87%
97%
99%
97%
100%
100%
90% 82%
83%N/A
86%
55%
87%
89% 69%
100%
100%71%
800
600
400
200
0
-200
E S T CGA TC G TC TG A TCG TC TA T A G TCG T CA TG T CG TA G TA TCA G T TCG T CA G TC G TC GA TCG T T CA G TC G T TC TG T TCG T CA TCGA TCGA T G TCA G TC G TC G TC TA T TGA T T CG TGA G TA G T CG TCGA
5
10
15
20
25
30
35
40
Control: No template control
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
H10 : T T AYGT T T T T AYGT T T T AT GYGGT AGYGGTAGAYGTTTYGGTTYGTYGTATTYGTTTTTTTYGTYGT/ GTTYGTTTYGGTTAATTAAYGAGYGTTYGYGTTTTTATTTTTATTTYGTGGAGTGGTYGGYGATAAGATGG
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/AN/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
30
20
10
0
-10
-20
-30
E S T C GA T CG TC TGA T C G TC TA TA G TCG T CA TG T CG TA G TA TC A G T T CG TCA G TCG TC GA T CG T T CA G TC G T TC TG T TC G TC A TCGA T CGA T G TCA G TC G T CG TC T A T TG A T TCG TGA G T A G TC G TCGA
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
105
110
115
120
125
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
4.1.3
ADS2501FS1 Validated by Pyrosequencing
Sequence to analyze:
TYGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGA
(Yellow highlights are the controls for bisulfite modification completion)
Histogram:
3
2
1
0
A
T
C
G
C
Pyrogram:
Control: Low methylated DNA
A11 : TYGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGA
T
G
T
G
C
5
T
G
10
3%
600
400
200
0
-200
E
S
A
T
C
G
C
Control: Medium methylated DNA
E11 : TYGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGA
T
G
T
G
5
C
T
G
T
G
T
G
T
G
10
45%
800
600
400
200
0
-200
E
S
A
T
C
G
C
Control: High methylated DNA
G11 : TYGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGA
T
G
T
G
5
C
10
94%
500
400
300
200
100
0
-100
E
S
A
T
C
G
C
Control: No template control
H11 : TYGTTTGGTGGTTGGTGAGATGGGTTAGAATGAATTAGA
T
G
T
G
5
C
10
N/A
0
-10
-20
-30
-40
E
S
A
T
C
G
C
5
T
G
T
G
C
10
ASSAY DESIGN REPORT
Assays/Assignment#
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Human HSD17B10/ADS2501 and ADS2502
4.2. PCR Bias Testing Using Pyrosequencing
Unmethylated DNA control and in vitro methylated DNA were mixed at different
ratios followed by bisulfite modification, PCR, and Pyrosequencing analysis. The
percent methylation obtained from the mixing study should be highly correlated
with expected methylation percentages with an r-square of 0.8 or higher.
4.3. Example of Quantitative Analysis
(in % Methylation)
ADS2502 FS1
ADS2502 FS2
From ATG
-171
-164
-142
-130
-122
-113
-107
-100
-95
-90
-87
-81
-72
-69
From TSS
-143
-136
-114
-102
-94
-85
-79
-72
-67
-62
-59
-53
-44
-41
GRCh36/hg18 53478188 53478181 53478159 53478147 53478139 53478130 53478124 53478117 53478112 53478107 53478104 53478098 53478089 53478086
GRCh37/hg19 53461463 53461456 53461434 53461422 53461414 53461405 53461399 53461392 53461387 53461382 53461379 53461373 53461364 53461361
Sample ID
CpG #1 CpG #2 CpG #3 CpG #4 CpG #5 CpG #6 CpG #7 CpG #8 CpG #9 CpG #10 CpG #11 CpG #12 CpG #13 CpG #14
0%
5.8
6.2
5.9
15.8
10.1
7.6
6.6
11.7
6.4
0.0
7.5
7.4
0.0
0.0
5%
1.4
1.3
5.5
2.5
1.3
0.8
0.0
3.4
6.0
1.9
5.9
1.8
2.6
1.9
10%
9.7
14.2
7.5
10.6
8.5
7.3
9.4
10.1
7.4
11.3
7.6
9.1
10.6
8.2
25%
12.4
14.6
9.5
10.6
14.8
13.3
14.3
16.5
14.8
18.6
15.4
10.3
7.0
9.7
50%
33.9
37.5
34.8
39.5
36.4
32.8
37.5
41.4
35.8
36.3
39.1
38.2
32.9
29.8
75%
46.2
49.4
38.6
45.8
51.0
45.5
44.2
38.3
51.8
53.3
49.2
48.3
41.9
39.3
100%
91.8
91.2
95.7
95.4
85.7
87.0
97.2
98.6
97.0
99.5
100.0
90.1
81.9
83.4
NTC
r-Square
0.9274
0.9416
0.8696
0.8772
0.9538
0.9337
0.9089
0.8520
0.9397
0.9539
0.9240
0.9363
0.9321
0.9245
ASSAY DESIGN REPORT
Assays/Assignment#
Page:
10(12)
Human HSD17B10/ADS2501 and ADS2502
5.
Protocols
5.1. Assay Template
5.1.1
Template
EpigenDx in-house control DNA was used for the assay development.
5.1.2
Bisulfite modification
Bisulfite modification was carried out using Zymo Research EZ Methylation kit
(Cat.#D5002 or D5004). 200 - 500 ng of sample DNA was used for bisulfite
modification followed by the PCR amplification.
5.2 PCR Protocol
5.2.1
Recommended PCR protocol
We recommend using HotStar Taq Polymerase Qiagen Cat.#203205. Additional
optimization is needed if different PCR system is used in analysis.
ADS2502
Component
10X PCR buffer (Contains 15mM
MgCl2)
25 mM MgCl2
10 mM dNTPs
10 M Fwd primer (ADS2502FP)
10 uM Rev primer (ADS2502RPB)
HotStar Taq Polymerase (5 U/l)
DNA
Water
Per 30l reaction
3 l (1x)
1.8 l (3.0 mM final conc.)
0.6 l (200 M of each)
0.6 l (6 pmol)
0.6 ul (6 pmol)
0.15 l (0.75 U)
1 µl of bisulfite treated DNA
Adjust to 30 µl
ADS2501
5.2.2
Component
Per 30l reaction
10X PCR buffer (Contains 15mM
MgCl2)
25 mM MgCl2
10 mM dNTPs
10 M Fwd primer (ADS2501FP)
10 uM Rev primer (ADS2501RPB)
HotStar Taq Polymerase (5 U/l)
DNA
Water
1.8 l (3.0 mM final conc.)
0.6 l (200 M of each)
0.6 l (6 pmol)
0.6 ul (6 pmol)
0.15 l (0.75 U)
1 µl of bisulfite treated DNA
Adjust to 30 µl
3 l (1x)
Recommended PCR cycling conditions:
ADS2502
95ºC 15 min; 45 x (95ºC 15s; 53ºC 30 s; 72ºC 30 s); 72ºC 5 min; 4C 
ADS2501
95ºC 15 min; 45 x (95ºC 15s; 60ºC 30 s; 72ºC 30 s); 72ºC 5 min; 4C 
ASSAY DESIGN REPORT
Assays/Assignment#
Human HSD17B10/ADS2501 and ADS2502
6.
Page:
11(12)
PyrosequencingTM analysis
The PSQ™96HS system should be used according to standard procedures with
the dispensation orders assigned for each assay as stated in this report.
Additional optimization may be needed if different analysis system is used.
6.1. Pyrosequencing Protocol:
Pyrosequencing is performed using PSQ 96HS system or PSQ 96HSA system.
The systems are equivalent to the PyroMark MD system. Pyrosequencing
analysis is performed as per manufactory’s recommended protocol with
modification:
1. Prepare master Binding Solution:
 Components for 1 reaction of binding solution
 2.2 l streptavidin
 40 l of 2X Binding Buffer
 25 l Milli-Q-water
2. Add 65 l of binding solution to each 15 l PCR sample.
3. Capture the PCR product as manufactory’s protocol.
4. Release the sepherose beads into annealing buffer containing 0.5 M of a
sequencing primer.
5. Anneal the sequencing primer to the template by heating the plate to 85 ºC
for 2 minutes.
6. Turn off the heating block and leave the Pyro plate on the heating block for
10 minutes.
7. Remove the Pyro plate from the heating block and allow the plate continuing
to cool for 5 minutes.
8. Run the Pyrosequencing as the manufactory’s instruction.
ASSAY DESIGN REPORT
Assays/Assignment#
Page:
Human HSD17B10/ADS2501 and ADS2502
12(12)
EpigenDx Assay Design Service (ADS)
General Terms and Conditions
1. Application
These general terms and conditions are applicable
to all of EpigenDx’s services. Every Customer
placing an order with EpigenDx accepts these
general conditions prior to placing the order.
2. Orders
Orders will only be acted upon if confirmed by a
written acceptance from Pyrosequencing.
3. Price
EpigenDx’s prices are quoted in US dollar and are
exclusive of VAT. Prices may be subject to change
at any time.
4. Delivery times
EpigenDx will use reasonable efforts to deliver
results within the period of time agreed with the
Customer.
However, agreed delivery times are not binding
and delays in delivery shall not give rise to
cancellation of the order.
5. Shipment of samples
When samples from the Customer are necessary in
order to perform the assay, the Customer shall
secure express approval from EpigenDx to ship the
samples to be analyzed by EpigenDx. The
Customer shall appropriately pack and label the
samples and pay for the shipment. EpigenDx shall
not be liable for loss or damage of the samples
during shipment, storage or use while performing
the ADS. After performing the services, EpigenDx
may, at its option, decide to return to the
Customer or to destroy any remaining samples.
EpigenDx reserves the right to refuse material that
is hazardous, controlled or prohibited by law.
6. Confidentiality
EpigenDx will use all reasonable endeavors to keep
in strict confidence any information and results,
related to an order by the Customer. Only the
Customer will be allowed to access such
information and results, except if EpigenDx is
required by law to disclose information or results.
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The Customer shall have the unrestricted right to
use any and all results, from their order. EpigenDx
shall own all right, title and interest in and to any
technology, methods, assay designs, know-how
and any inventions used by EpigenDx to perform
the service for the Customer.
8. Limited warranty
EpigenDx shall use all reasonable efforts to
perform the services with utmost care and skill.
EpigenDx makes no other Warranty, express or
implied, including any warranty of merchantability,
title, or fitness for a particular use.
In particular, EpigenDx does not warrant that the
use of results will not infringe intellectual property
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9. Limitation of liability
After delivery of the results and material to the
Customer, EpigenDx assumes no further liability
than that provided under Article 8.
In no event shall EpigenDx be held liable for any
damages arising from loss of earnings or any other
loss arising directly or indirectly from the use of the
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The Customer agrees to assume any and all
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of the results and material.
10. Payment
Invoices are due and payable within thirty calendar
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EpigenDx shall have the unilateral right to cancel
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12. Applicable Law
This agreement will be governed and interpreted in
accordance with US law.
For research use only. Not intended for diagnostic purposes.
These mutations may be covered by a patent or patent applications. This protocol is made available for scientific research
only and in no way confers the rights to perform this assay for commercial purposes or profit.
© Copyright 2006 EpigenDx.
Pyrosequencing, PSQ, Pyrogram and
are trademarks owned by Qiagen. Pyrosequencing technology
is covered by patents and patent applications owned by Qiagen.