Pre-amplification multiplex results for bisulfite DNA samples. (A) A flow diagram outlining the different conditions examined with respect to multiplexability, pooling, and exonuclease treatment. (B) The results of different conditions after 15 cycles of multiplex pre-amplification. Forty-eight primer pairs were assessed for the multiplexability in a pre-amplification reaction. All samples had the same amount of input DNA. Lane 1: A positive control involving singleplex PCR reaction of an individual primer pair, with the same DNA template amount as used in the pre-amplification. Lane 2: Eight-plex pre-amplification reaction and ExoSAP-IT treatment of individual pre-amp reactions, followed by pooling and singleplex amplification (as illustrated in the upper panel of A). Lane 3: Results of three different 8-plex reactions pooled together first then ExoSAP-IT treatment of the combined pool, for a total of 24 amplicons in the ExoSAP-IT treatment, followed by singleplex amplification of a primer pair (as illustrated in the lower panel of A). Lane 4: Twenty-four-plex pre-amplification results. (C) The effect of ExoSAP-IT treatment on bisulfite libraries, as compared to gDNA amplicons. The arrow indicates where primers migrate on the gel. Lane 1: Pool of 48 amplicons prior to barcoding PCR. Lane 2: Sample library after barcoding PCR. Primers are visible at the bottom of the lane. Lane 3: Sample library after ExoSAP-IT treatment at 37°C. Lane 4: Sample library after ExoSAP-IT treatment at 37°C, followed by heat inactivation of the ExoSAP-IT at 80°C. Note the higher molecular weight smear in the methylation library, which is not observed with gDNA amplicons. Lane 5: Sample library cycled at 37°C, followed by 80°C heat denaturation step, but with no ExoSAP-IT.