Supplementary Materials for

Identifying DNA methylation biomarkers for non-endoscopic detection

of Barrett’s esophagus

Helen R. Moinova, Thomas LaFramboise, James D. Lutterbaugh,

Apoorva Krishna Chandar, John Dumot, Ashley Faulx, Wendy Brock,

Omar De la Cruz Cabrera, Kishore Guda, Jill S. Barnholtz-Sloan, Prasad G. Iyer,

Marcia I. Canto, Jean S. Wang, Nicholas J. Shaheen, Prashanti N. Thota,

Joseph E. Willis,* Amitabh Chak,* Sanford D. Markowitz*

*Corresponding author. Email: SXM10@cwru.edu (S.D.M.); josephe.willis@uhhospitals.org (J.E.W.);

amitabh.chak@uhhospitals.org (A.C.)

Published 17 January 2018, Sci. Transl. Med. 10, eaao5848 (2018)

DOI: 10.1126/scitranslmed.aao5848

This PDF file includes:

Fig. S1. Flowchart of study analyses.

Fig. S2. Comparison of methylation of individual CpGs versus CpG patches in

discriminating esophageal lesions.

Fig. S3. Comparing VIM and CCNA1 expression versus methylation.

Fig. S4. Morphology of touch preps from balloon brushings of three intact porcine

esophagus samples from esophagogastric organ explants.

Table S1. Location of 26 differentially methylated patches identified by RRBS

comparison of normal esophageal squamous mucosa versus Barrett’s lesions.

Table S2. Demographic characteristics of training and validation esophageal

brushing populations.

Table S3. mVIM and mCCNA1 performance in training and validation

esophageal brushing samples.

Table S4. Comparison of sensitivities of mVIM plus mCCNA1 versus mVIM or

mCCNA1, all at equal specificities.

Table S5. Influence of smoking on VIM and CCNA1 methylation in proximal

versus distal esophagus.

Table S6. Participant evaluation of the non-endoscopic balloon sampling of the

esophagus.

Table S7. Demographic characteristics of subjects in non-endoscopic balloon

study.

www.sciencetranslationalmedicine.org/cgi/content/full/10/424/eaao5848/DC1

Table S8. Methylation in post-ablation subjects.

Table S9. Post-examination questionnaire.

Table S10. Bisulfite-specific methylation-independent PCR primer sequences.

Table S11. Index tags for bisulfite-specific methylation-independent PCR primer

sequences.

References (33–66)

Fig. S1. Flowchart of study analyses. Sample abbreviations are as follows: N Sq, normal

squamous epithelium from the proximal esophagus; Control GEJ, unaffected controls brushed at

the gastroesophageal junction; NDBE, nondysplastic Barrett’s esophagus; ND SSBE,

Balloon set36 Normal Controls

13 ND SSBE18 ND LSBE

6 LGD4 HGD

8 EAC/JCA

Device test and cutoff validation

Brushing validation set30 Control GEJ

19 ND SSBE23 ND LSBE

26 LGD13 HGD

38 EAC/JCA

Cut-off validation

Brushing training set62 Control GEJ

12 ND SSBE19 ND LSBE

8 LGD10 HGD

62 EAC/JCA

Establishment of assay cut-offs

Biopsy Validation74 N Sq16 NDBE19 HGD

68 EAC/JCA

NGS assay development, patch validation

Reduced Representation Bisulfite Sequencing26 N Sq26 EAC14 BE

5 EAC cell lines

Discovery of differentially methylated patches

nondysplastic short segment Barrett’s esophagus of 1-3 cm; ND LSBE, nondysplastic long-

segment Barrett’s esophagus of > 3 cm; LGD, Barret’s esophagus with low-grade dysplasia;

HGD, Barrett’s esophagus with high-grade dysplasia; EAC/JCA, esophageal adenocarcinoma

and/or junctional cancer of the esophagus.

B A

D E

C

Fig. S2. Comparison of methylation of individual CpGs versus CpG patches in

discriminating esophageal lesions. (A, B) Heat map of the methylation at each individual CpG

in the CCNA1 (A) and VIM (B) methylation patches as assayed in the validation brushing sample

set (detailed in table S3). 21 CpGs are shown for mCCNA1, and 10 CpGs for mVIM. Red color

corresponds to most methylated, and blue color corresponds to least methylated. Normal,

brushings of the GE junction region from endoscopically-normal controls; EAC, esophageal

adenocarcinoma and/or junctional cancer of the esophagus; HGD, Barrett’s esophagus with high-

grade dysplasia; LGD, Barret’s esophagus with low-grade dysplasia; ND LSBE, nondysplastic

Barrett’s esophagus of > 3 cm; SSBE, nondysplastic short segment Barrett’s esophagus of 1-3

cm. (C, D), Box-and-whiskers plots comparing methylation at each individual CpG for CCNA1

(C) and VIM (D) in cases (blue) versus controls (red). Cases are all EAC and BE samples with or

without dysplasia. Controls are brushings of the GE junction region from endoscopically-normal

control subjects. One individual control sample (indicated by °) is an outlier with increased

methylation in all comparisons. (E) Box-and-whiskers plots demonstrating discrimination of

F G

cases (red) from controls (blue) on comparison of the percent of methylated reads detected in

bisulfite sequencing analysis across the full mVIM and mCCNA1 target patches (percent of

patch methylation). (F, G) Graphs show the comparison of the sensitivities for detection of cases

by assay of methylation across patches (blue circles) versus at individual CpGs (orange circles),

determined at the same specificity for all the assays. To assess reproducibility of performance,

comparisons were determined in both the validation set of brushing samples (x-axis) and the

balloon samples (y-axis). When compared at the same specificity, the patch-based analyses

exhibit superior and more reproducible sensitivity than do assays of individual CpGs. (F)

mCCNA1 and (G) mVIM. assay cutoffs for individual CpGs were selected to match the

specificity of the parent patch when tested in the same sample set (table S3 for brushings and

Table 2 for balloons).

Fig. S3. Comparing VIM and CCNA1 expression versus methylation. A, CCNA1, B, VIM.

Normalized mRNA expression was determined by qPCR, relative to the housekeeping gene

B2M. Normal testis total RNA (Clontech) was used as a positive control for CCNA1 expression.

CCNA1 expression in normal squamous and EAC cell lines was <10-3 of the control. V364, a cell

line from an undifferentiated abdominal tumor of unknown origin, was a positive control for

VIM expression. Expression of VIM in normal squamous and esophageal cell lines (other than

FLO1) was < 10-2 of the control. Methylation was measured by NGS sequencing. Blue:

methylation less than 1%. Gray: methylation of 1-4%. Orange: methylation of 93-97%. Red:

methylation of >97%. No VIM or CCNA1 expression was detected in normal squamous

epithelium, in which both loci are unmethylated. CCNA1 is also not detected in EAC cell lines,

in which the locus is methylated. VIM expression was detected in one EAC cell line (FLO1)

that is unmethylated, suggesting the possibility that methylation does serve a role in

transcriptional repression of VIM in esophageal cancers but not in the normal esophagus.

B A

Methylation:

Fig. S4. Morphology of touch preps from balloon brushings of three intact porcine

esophagus samples from esophagogastric organ explants. Morphology demonstrates sampling

of only an epithelial cell population.

Table S1. Location of 26 differentially methylated patches identified by RRBS comparison of normal

esophageal squamous mucosa versus Barrett’s lesions. The nearest neighbor gene is listed for each differentially

methylated patch, and citations are noted for any prior findings of methylation in BE, EAC, or other cancers.

Amplicon name

Number of CpGs in amplicon

Amplicon coordinates for validated amplicons (hg19)

Nearby Gene

Literature evidence of methylation in BE/EAC

Literature evidence of methylation-- other cancers

SqBE 1 12 chr1:4,714,435-4,714,573

AJAP1 No

non-small cell lung cancer(33), oral squamous cell carcinoma (34), hepatocellular carcinoma (35), glioma (36), glioblastoma (37), gastric cancer (38), endometrial atypical hyperplasia (39), cervical cancer (40)

SqBE 2 16 chr1:170630519-170630783

PRRX1 No bladder cancer (41), Aldosterone-producing adenomas of the adrenal gland (42)

SqBE 4 25 chr4:6,201,296-6,201,535

JAKMIP1 No No

SqBE 5 15 Chr:5:1883179-1883413

IRX4 No oropharyngeal squamous cell carcinoma (43)

SqBE 7 27 chr6:125283738-125284007

RNF217 STL

No No

SqBE 8 20 chr7:31,092,274-31,092,480

ADCYAP1R1 No No

SqBE 9 31 chr:8:68864745-68865014

PREX2 No No

SqBE 10 10 chr9:132382809-132382957

C9ORF50 No Colorectal cancer (44)

SqBE 11-1

17 chr10:7708626-7708857

ITIH5 No Colon cancer (45), bladder cancer (46), breast cancer (47), cervical cancer (48), tongue squamous cell carcinoma (49)

SqBE 11-2

10 chr10:7708818-7708933

SqBE 12 11 chr10:60,273,232-60,273,431

BICC1 No No

SqBE 13 19 chr10:79397312-79397500

KCNMA1 No gastric cancer (50), prostate cancer (51)

SqBE 14-1

8 chr10:133108916-133109175

TCERG1L No colon cancer (52), Crohn's Disease (53)

SqBE 14-2

22 chr10:133109090-133109361

SqBE 15 11 chr11:57250430-57250616

SLC43A1 No No

SqBE 16-1

26 chr11:110582474-110582722

ARHGAP20 No prostate cancer (54)

SqBE 16-2

26 chr11:110582697-110582905

SqBE 17-1

22 chr12:99288803-99288998

ANKS1B No No

SqBE 17-2

7 chr12:99289167-99289280

SqBE 18 21 chr13:37005856- CCNA1 No Oral cavity squamous cell carcinoma (55), cervical cancer (56), breast cancer (57),

37006031 low grade papillary urothelial cell carcinoma (58), colorectal cancer (59)

SqBE 21 13 chr14:85,997,844-85,998,118

FLRT2 No Breast cancer (60), lung cancer (61), prostate cancer (62)

SqBE 22-1

9 chr15:41793974-41794125

ITPKA No Lung and breast cancer (63, 64)

SqBE 22-2

20 chr15:41793818-41794001

SqBE 23 10 chr15:79383659-79383890

RASGRF1 No gastric cancer (65), breast cancer (66)

SqBE 25-1

8 chr21:40,357,592-40,357,815

LINC01700 No No

SqBE 25-2

6 chr21:40,357,788-40,358,053

Training set

Validation set

Cases

(n=111)

Controls

(n=62)

P value cases vs

controls

Cases

(n=119)

Controls

(n=30)

P value cases vs

controls

Age mean (st dev) 65.8

(11.8)

53.8

(15.9)

p <

0.0001

68.3

(11.0)

61.1

(16.4)

p =

0.0044

Race

White 103 40 W vs B

p <

0.000001

117 21 W vs B

p =

0.0001

Black 4 22 2 7

Other 4 0 0 2

Gender

Male 95 27 p <

0.000001

87 12 p =

0.001019 Female 16 35 32 18

Smoking

history

Former or

current smoker 70 32

p = 0.15

71 9

p = 0.042

Non-smoker 41 30 45 15

Unknown 3 6

Table S2. Demographic characteristics of training and validation esophageal brushing

populations. Continuous variables (age) were compared by T-test and categorical variables by

Fisher’s exact test.

Table S3. mVIM and mCCNA1 performance in training and validation esophageal

brushing samples. VIM and CCNA1 gene methylation was assayed in DNA samples from

cytology brushings of the distal esophagus from: Unaffected controls brushed at the gastro-

esophageal junction (control GEJ); and from cases of nondysplastic Barrett’s esophagus

(NDBE), further subclassified as short-segment Barrett’s esophagus of 1-3 cm (SSBE) or long-

segment Barrett’s esophagus of > 3 cm (LSBE); Barret’s esophagus with low-grade dysplasia

(LGD); Barrett’s esophagus with high-grade dysplasia (HGD); esophageal adenocarcinoma

and/or junctional cancer of the esophagus (EAC). Samples were scored as VIM methylated for

mVIM >1.05% and as CCNA1 methylated for mCCNA1 >3.12% (using ROC defined cutpoints

from Fig. 3A, B). Cases were positive for the panel of mCCNA1 plus mVIM if either marker

tested positive. Controls were negative for the panel when both mCCNA1 and mVIM were

Training population Validation population

mVIM mCCNA1 Either mVIM

or mCCNA1 mVIM mCCNA1

Either mVIM

or mCCNA1

% n % n % n % n % n % n

Specificity

control GEJ 93.2% 59 98.4% 61 91.4% 58 92.6% 27 92.9% 28 88.5% 26

Sensitivity all

cases 90.7% 107 90.7% 108 95.5% 111 91.5% 117 89.6% 115 94.1% 118

Sensitivity

NDBE 90.0% 30 79.3% 29 90.3% 31 92.7% 41 80.0% 40 92.7% 41

SSBE 91.7% 12 83.3% 12 91.7% 12 84.2% 19 72.2% 18 84.2% 19

LSBE 88.9% 18 76.5% 17 89.5% 19 100% 22 86.4% 22 100% 22

Sensitivity

dysplastic BE 100% 17 94.4% 18 100% 18 87.2% 39 94.6% 37 94.9% 39

LGD 100% 7 87.5% 8 100% 8 92.3% 26 91.7% 24 92.3% 26

HGD 100% 10 100.% 10 100% 10 76.9% 13 100% 13 100% 13

Sensitivity

EAC 88.3% 60 95.1% 61 96.8% 62 94.6% 37 94.7% 38 94.7% 38

negative. Controls with one negative marker and one marker with assay failure were excluded.

Entries indicate percent sensitivity or specificity (%) and total number of individuals tested (n).

Either mVIM or

mCCNA1 mVIM

mCCNA1

% n % n % n

Specificity control GEJ 90.5% 84 90.7% 86 91.0% 89

Sensitivity all cases 94.8% 229 91.1% 224 91.5% 223

Sensitivity all NDBE 91.7% 72 93.1% 71 80.3% 69

Sensitivity SSBE 87.1% 31 87.5% 31 74.2% 30

Sensitivity LSBE 95.1% 41 97.5% 40 85.0% 39

Sensitivity all dysplastic BE 96.5% 57 89.3% 56 96.3% 55

Sensitivity LGD 94.1% 34 90.9% 33 93.5% 32

Sensitivity HGD 100.0% 23 87.0% 23 100.0% 23

Sensitivity EAC 96.0% 100 90.7% 97 94.9% 99

Table S4. Comparison of sensitivities of mVIM plus mCCNA1 versus mVIM or mCCNA1,

all at equal specificities. Shown is analysis of all esophageal brushings from combined training

and validation populations. Disease categories and samples are as in table S3. Sensitivity of the

panel of mVIM plus mCCNA1 (either mVIM or mCCNA1) is shown, as in Table 2, at a

specificity of 90.5%, with mVIM positive when >1.05% and mCCNA1 positive when >3.12%.

Comparison is shown versus the sensitivities of mVIM alone (mVIM) when adjusted to

specificity of 90.7% (mVIM positive when >1.0%) and versus mCCNA1 alone (mCCNA1)

when adjusted to specificity of 91% (mCCNA1 positive when >1.6%). Entries indicate percent

sensitivity or specificity (%) and total number of individuals tested (n).

Gene Comparison groups Median

methylation

Range (minimum,

maximum)

Sample

size

Two-tailed

probability

(Mann-

Whitney)

VIM

Proximal normal sq-

nonsmoker

0.1% (0%, 7.4%) 71

P = 0.0155a

Proximal normal sq-smoker 0.1% (0%, 69.2%) 102

CCNA1

Proximal normal sq-

nonsmoker

0.1% (0%, 2.2%) 69

P = 0.0095a

Proximal normal sq-smoker 0.1% (0%, 67.6%)

VIM Control GEJ-nonsmoker 0.1% (0%, 50.0%) 41

P = 0.2506 Control GEJ-smoker 0.1% (0%, 7.0%) 39

VIM Cases (BE/EAC)-nonsmoker 29.6% (0%, 87.8%) 83

P = 0.2999 Cases (BE/EAC)-smoker 49.8% (0%, 93.5%) 139

CCNA1 Control GEJ-nonsmoker 0.1% (0%, 3.1%) 42

P = 0.9271 Control GEJ-smoker 0.1% (0%, 22.7%) 41

CCNA1 Cases (BE/EAC)-nonsmoker 45.4% (0%, 89.0%) 83

P = 0.1356 Cases (BE/EAC)-smoker 52.4% (0%, 91.9%) 138

Table S5. Influence of smoking on VIM and CCNA1 methylation in proximal versus distal

esophagus. Upper cells: comparisons of proximal normal squamous mucosa in smokers versus

non-smokers (denoted by superscripta) correspond to analyses presented in Fig. 4. Lower cells:

comparisons of distal esophagus in smokers versus non-smokers. In contrast to the significant

difference in methylation detected in brushings of the proximal esophagus of smokers versus

non-smokers, the lower cells show no significant difference in VIM or CCNA1 methylation

between smokers and non-smokers when assessed in brushings at the gastroesophageal junction

(GEJ) or distal esophageal lesions (BE/EAC).

Controls: individuals with finding of GERD, erosive esophagitis, or no pathology, brushed at the

GEJ. Cases: brushings of lesions from individuals with BE or cancer. Proximal normal sq:

Brushings of normal-appearing squamous mucosa from proximal esophagus, obtained from

individuals with or without a neoplastic lesion at the GEJ. Individuals were classified as smokers

if they had current or past history of smoking. Nonsmoker classification was applied to

individuals without any history of smoking. Mann-Whitney rank sum test was used for between-

group comparisons.

Scoring

category

10-point Response Scale, percentage (n/total)

1-2 (low discomfort) 3-8 (intermediate discomfort) 9-10 (high discomfort)

% n/total % n/total % n/total

Anxiety 75% 96/128 24% 31/128 1% 1/128

Choking 82% 105/128 17% 22/128 1% 1/128

Gagging 52% 66/128 45% 58/128 3% 4/128

Pain 95% 121/128 5% 7/128 0% 0/128

Overall

tolerance 72% 92/128 28% 36/128 0% 0/128

Scoring

category

5-point Response Scale, percentage (n/total)

4-5 (affirmative) 3 (unsure) 1-2 (negative)

% n/total % n/total % n/total

Would

recommend to

others

95% 122/128 3% 4/128 2% 2/128

Would repeat

procedure 93% 118/128 5% 6/128 3% 4/128

Table S6. Participant evaluation of the non-endoscopic balloon sampling of the esophagus.

Study participants evaluated the tolerability of the balloon device sampling of the esophagus by

completing a post-exam questionnaire (table S9). Responses to each question were scored on 10-

point or 5-point Likert scales and analyzed by categories as specified in the table. Entries

indicate percent of respondents in each category (%), absolute number of respondents in each

category (n), and total number of respondents.

Cases (n=50) Controls (n=36) P value cases vs

controls

Age mean (st dev) 68.5 (11.0) 55.6 (12.1) p<0.0001

Race

White, n (%) 46 (92%) 24 (67%)

W vs B

P = 0.005834

Black, n (%) 2 (4%) 9 (25%)

Other, n (%) 2 (4%) 3 (8%)

Gender

Male, n (%) 42 (84%) 16 (44%)

P = 0.000164 Female, n (%)

8 (16%) 20 (56%)

Smoking

history

Former or current

smoker, n (%) 32 (64%) 17 (47%)

P = 0.121581 Non-smoker, n (%) 17 (34%) 19 (53%)

Unknown, n (%) 1 (2%) 0 (0%)

Table S7. Demographic characteristics of subjects in non-endoscopic balloon study.

Continuous variables (age) were compared by t-test and categorical variables by Fisher’s exact

test. Percentages do not always add up to 100 due to rounding.

mVIM mCCNA1 Either mVIM of

mCCNA1

Sample

counts

Specificity for

evaluable normal

controls

91.7% 100.0% 91.7% 36

Specificity for

excluded post-ablation

endoscopically-

normal subjects

65.2% 91.3% 60.9% 23

P-value P = 0.016647 P = 0.147867 P = 0.007019

Table S8. Methylation in post-ablation subjects. Shown is specificity of mVIM and mCCNA1

in evaluable normal controls, defined as subjects without current or past BE, versus

endoscopically-normal subjects who have had ablation of dysplastic BE in the past and were

therefore excluded from study analyses. Evaluable normal controls: Individuals with no prior

history of esophageal neoplasia or ablation procedures. Post-ablation subjects: Individuals with

prior history of ablation procedure to remove dysplastic BE, but who show no residual BE on

endoscopic examination at the time of balloon sample collection. Post-ablation subjects showed

a significantly higher rate of mVIM and mCCNA1 detected methylation than did normal

controls, suggesting that residual molecularly abnormal clones of esophageal mucosa remain

after the ablation procedure. Fisher’s exact 2-tailed test was used for between-group

comparisons.

PROCEDURE TOLERABILITY AND ACCEPTABILITY

(Complete after the capsule balloon test)

Next, we want to ask you some questions about YOUR EXPERIENCE with the Capsule

Balloon procedure. Please mark only one number for each question by filling in the circle

completely.

1. Your level of anxiety, nervousness or worried feelings that you experienced about having

the capsule test.

No worries I was terrified

1 2 3 4 5 6 7 8 9 10

2. The level of pain that you experienced with the procedure.

No pain Most severe pain

1 2 3 4 5 6 7 8 9 10

3. The level of gagging or retching that you experienced with the procedure.

No gagging Most severe gagging

1 2 3 4 5 6 7 8 9 10

4. The level of choking you experienced during the procedure.

No choking Most severe choking

1 2 3 4 5 6 7 8 9 10

5. The level of overall tolerance of the procedure.

No difficulty Severely intolerable

1 2 3 4 5 6 7 8 9 10

6. We would appreciate written comments regarding what was the most difficult part of

the capsule balloon procedure:

Acceptability Of Screening Test

Please indicate the extent to which you agree or disagree with each of the following statements. Use a scale

of 1 to 5, with 5 being Strongly Agree and 1 being Strongly Disagree. If an item is not related to your care

choose N/A.

Strongly

Disagree

Somewhat

Disagree Neutral

Somewhat

Agree

Strongly

Agree N/A

(1) (2) (3) (4) (5)

I would undergo the balloon test again if needed for

further care. O O O O O O

I would recommend this screening balloon test to

family and friends. O O O O O O

Table S9. Post-examination questionnaire. Questionnaires were completed after esophageal

sampling by the balloon device. The summary of survey results is presented in table S6.

Gene name

Primer

name Primer Sequence

Number of

CpGs in

amplicon

Number of

methylated CpGs

required for a

methylation

positive read

VIM

F-M CTCGTCCTCCTACCGCAAAATATTC

10 8 F-U CTCATCCTCCTACCACAAAATATTC

R-M GTTGTTTAGGTTGTAGGTGCGGG

R-U GTTGTTTAGGTTGTAGGTGTGGG

CCNA1

F-M GCGATTGTATTTGGGGTAGTTT

21 16 F-U GTGATTGTATTTGGGGTAGTTT

R-M CCCGCTCCTAAAAACCCTAACT

R-U CCCACTCCTAAAAACCCTAACT

Table S10. Bisulfite-specific methylation-independent PCR primer sequences. Forward (F)

and reverse (R) PCR primers were constructed as a mixture of primers against the bisulfite

converted products of fully methylated (M) or fully unmethylated (U) templates, and were used

to amplify a differentially-methylated region of the VIM exon 1 CpG island (31) or CCNA1.

Index tag number Index tag sequence

1 TGTAGAT

2 GACCTGT

3 AAGACGC

4 TTCAATG

5 CTTCCAG

6 AGCTTCA

7 TATGAAA

8 GTGTTAG

9 CCTTGTA

10 ATGAAAT

11 TAGAGAA

12 AGACGAG

13 GTCGTGG

14 CACCACG

15 CCACAAA

16 TTATAGC

17 GGCAAGC

18 TTTACCG

19 TCAGGGG

20 TGCTCTT

21 TCTGCCT

22 GCAGCCC

23 CGCGGAA

24 ACATCCG

25 ACTAAAA

26 AGTTACT

27 GTAGGGC

28 GAAGAAC

29 AGCGAGT

30 CGGTTGA

31 GCCTAGT

32 CTGCTCC

33 TGGCTGG

34 CGGTCAC

35 TGAGCAC

36 AGTATGC

37 GCGCCTC

38 CCTCAGC

39 CAAACTC

40 ATTCGCT

41 AGGGCAG

42 CTCCGTC

43 GGTACTG

44 TTGTCCC

45 ATACACA

46 GGCATAG

47 ATATTAC

48 GGGAATA

49 GATTTAA

50 GTCCTAA

51 TGGACCT

52 GAGTAGG

53 TATAGCC

54 TGCCGGT

55 ATGCCAA

56 AAGCCTG

57 ATCTGAA

58 TAACTGA

59 CCGTAAT

60 ACCCACC

61 CGTCGAC

62 ACCCTAT

63 GCAGATT

64 GGAGTCA

65 ACCGCTT

66 TTGTGGA

67 CCCAAAG

68 CCATGCC

69 GCAACGT

70 TAAGGTC

71 TCCGCGA

72 CCCGTCT

73 AAAACCT

74 GAACGCA

75 CTAAGCT

76 CGTTATG

77 ATGAGCG

78 TAGGCGT

79 CTAGGTA

80 ATAACAG

81 GACCGAG

82 CGTCTCG

83 TCTCATA

84 AACGAAG

85 TTTCGAA

86 GTGTCTA

87 AGCGGTC

88 TCACCTT

89 GAGCACT

90 GAGACAG

91 CTACCGT

92 ACAGGCA

93 TGATTCT

94 CGGGTTC

95 TGCGCCG

96 TCGCAAG

Table S11. Index tags for bisulfite-specific methylation-independent PCR primer

sequences. The 7-nucleotide tags listed in this table were added to the 5’ end of both forward

and reverse primer sequences listed in table S10 to create 96 different patient-specific indexed

primers. This approach allows barcoding each patient sample in the first round of PCR, before

PCR products are pooled to create the NGS sequencing library.