Table 1 Summary of singleplex bisulfite PCR results where the modified base is located in either a single primer or in both primers.

Forward Primer	Reverse Primer	Which primer was modified	Deoxyinosine	Mixed-base	5-nitroindole	Dspacer	CpG-free control
M201	M202	Neither	—	—	—	—	Positive
M203	M204	Neither	—	—	—	—	Positive
M205	M206	Neither	—	—	—	—	Positive
M209	M210	Forward	Positive	Positive	No amplification	No amplification	—
M213	M214	Forward	Positive	Positive	Positive	Positive	—
M215	M216	Forward	Positive	Positive	Positive	Positive	—
M217	M218	Both	Positive	Positive	No amplification^#	No amplification^#	—
M219	M220	Both	Positive	Positive	No amplification	No amplification	—
M221	M222	Both	Positive	Positive	No amplification	No amplification	—

^*modifications are located in either the ‘forward’ or ‘both’ the forward and reverse primers. In each state, the modification is located near the 5′ end of the primer(s). In ‘CpG-free’ control primers, ‘Neither’ implied that that both the forward and reverse primers have no modifications. Primer sequences are supplied in SuppTable 1. ^#Resultant product on gel was unclear and therefore excluded from further analysis.

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