Table 2:

Amplification primers for pyrosequencing were designed with a biotin-label on either the forward (F) or reverse (R) primer and used to amplify bisulfite converted DNA on the Applied Biosystems 7900HT. The sequence of each primer is listed 5’ to 3’ along with the amplicon length and the CpG sites it amplifies.

CpG Site	Amplification Primer	Amplicon Length	Pyrosequencing Primer
1 to 5	F: 5’ TTTTAGGTGAAGTTGTTGTAGGT 3’	108	F: 5’ AGGTAGAGATGGGTTTTGTA 3’: 1–3
	R: 5’ /5biosg/ TCCCRTACAATTTCCAAAATTT 3’		R: 5’ TATATAGGGAATAA 3’: 4–5
6 to 11	F: 5’ TTAGGAATTTAGGTTAGAATTTAGGTTAAG 3’	143	F: 5’ AGGTTAAGTTTGTTATAG 3’: 6–7
	R: 5 ‘/5biosg/CCTATCAAAATAAAAAAACCAAAAC 3’		R: 5’ ATTAATTGGGGTTTAGT 3’: 8–11
12 to 25	Not Targeted	?	Not Targeted
26 to 34 (26–27; 30–34 not targeted)	F: 5’ /5biosg/ TGTATTTATTTTTTTAATTTTTAGG 3’	156	F: 5’ TTTTTAGGTTYGGGTTTGGTG 3’: 28–29
	R:5’/5biosg/ CCCTCCCTCTACAATACAAAA 3’
35 to 43	F: 5’ /5-biosg/ TTGGTTTTTGTATTGTAGAGGG 3’	135	F: 5’ GGTTTAATTTTGTTTT 3’: 35–40
	R: 5’ /5’biosg/ AAACTCTTAAACCCCCAACTAA 3’		R: 5’ GTTTTYGGTYGGTTYGATTT 3’: 41–43
44 to 49	F: 5’ AGTTGGGGGTTTAAGAGTTTTGG 3’	288	F: 5’ GGTTTTGGTAGTGGTATAGT 3’: 44–46
	R: 5’ /5’biosg/ AAAACCCTTCCCAAAAAAATCC 3’		R: 5’ GGTGTTGGGTTTTATTTATTTAG 3’: 47–48
51	F: 5’/ 5biosg/ GGGAAGGGTTTTTTATTAAGAG 3’	?	F: 5’ AATAAAAAACCTAAAATAAC 3’: 51
	R: AACCCCATACAACTACAAATATAC 3’
52 to 53	F: 5’ ATTTGGATATATAAGGTAAGGTGGAA 3’	92	F: 5’ TGATATTGTGGAGATTGG 3’: 52–53
	R: 5’ /5biosg/ ACTACCCCTTTAATTCTCATTCC 3’
54 to 63	F: 5’GATTGGGCGCGTAGTATTTTTAG 3’	215	5’ TGAGAATTAAAGGGGTAGT 3’: 54–57
	R: 5’ /5’biosg/AACTCTATTCCCCACTAAACCAAC 3’		5’ TTYGTGYGTTTTYGAGTGYGTG 3’: 58–61
64 to 65	F: 5’ GGGAAGAGATTTTATTTAAAGAGA 3’	145	F: 5’ TAAAAGTAAATAGAGGGTAG 3’: 64
	R: 5’ /5biosg/ AATCAACTCTCTCTTTTCACCAT 3’		R: 5’ TAAGTTTGATTATATGATTTTGAT 3’: 65
66 to 67	F: 5’ /5biosg/ ATGGTGAAAAGAGAGAGTTGATT 3’	112	F: 5’ CATTTACTCTATAAATAC 3’: 66
	R: 5’ AAATTCCCTCTACCTCCTTTTAA 3’		R: 5’ CTCTACCTCCTTTTAAAC 3’: 67
68 to 70	F: 5’ /5biosg/ GGAATGTGATGTTAGGATTGTA 3’	?	F: 5’ CCAACTCCTCTTAAATTC 3’: 68–70
	R: 5’ CTTTACCTTTCATCTACAAAAA 3’
71 to 73 (71 not targeted)	F: 5’ /5biosg/ TTTAAGAAGGATGATGGTTTTT 3’	101	F: 5’ AAAATAATTACCAAAATC 3’: 72
	R: 5’ TCATCTACAAAAAACAAAACAA 3’		R: 5’ TCATCTACAAAAAACAAAACAAC 3’: 73
74 to 78	F: 5’TTGTTTTGTTTTTTGTAGATGA 3’	118	F: 5’ GTTAAGGTTTATTATAGTTAG 3’ : 74–76
	R: 5’/5biosg/ CTTTTATCAAAACCCTCCTC 3’		R: 5’ TTAGTGGTTTTAGGAATA 3’: 77–78
79 to 84	F: 5’ GAGGAGGGTTTTGATAAAAG 3’	162	F: 5’ GGTTTTGATAAAAGATGG 3’ : 79–80
	R: 5’ /5biosg/ AAAAACCAAATACCTACAAAAAT 3’		R: 5’ GAGTATATTGTTGATAAG 3’: 81
85 to 87	F: 5’/5biosg/ CGATTTTTATTGATATGTAGTGGC 3’	?	F: 5’ ACRACCAAATACCACACCAC 3’: 85
	R: TTAACAAAAAATCCCAACTCCAAC 3’		R: 5’ AACTCCAACAAATCCCC 3’: 86–87