Figure 3: Uniqueness of primers in the genome as a predictor of PCR fidelity.

To determine if uniqueness of primers in the genome can be used to predict the fidelity of PCR, primers were mapped to two different reference genomes, both of which were bisulfite converted prior to mapping. (a) Primer-to-human-genome (hg19) mapping. Distribution of the number of primer-to-genome matches when mapping with Bowtie1 in single-end mode against hg19, as a function of PCR product as visualized by DNA gel electrophoresis. The box-and-whisker plot shows a correlation between the numbers of primer-to-genome matches to number of gel bands. (b) Primer-to-Repbase mapping. The stacked bar chart shows an increase in the number of exact primer-to-reference-matches when mapping with Bowtie1 in single-end mode. Overall the number exact primer-to-reference-matches increased with the number of PCR products visualized on a DNA gel (i.e. single verses multiple products). (c) Visualisation of amplicons which produced different sized gel bands. Of the 82 primer pairs which mapped uniquely to the hg19 genome (i), 76 produced a single PCR product. Of the 10 primer pairs which mapped to multiple loci (ii), only one produced a single PCR product as visualized by DNA gel electrophoresis; whereas all primer pairs which mapped multiple times with a difference in amplicon size between the smallest and largest products of at least 10 base pairs can be seen on gel as multiple bands. Although the target amplicon of approximately 120 base pairs appeared to be present in all the gel images (iii), the presence of other PCR products in the gel was hypothesised to be the amplification of repetitive sequences as indicated by alignment of primers against the Repbase (homo sapian) repetitive regions library.