vRLGS spot candidate identification and confirmation. Human NotI-EcoRV-HinfI profiles zoomed into section 3D. Identified spots are labeled on the lower panels for A) and B), with the upper panels showing the overlap of the actual and virtual spots of interest – 62 and 68 – circled. All other labeled spots were previously identified. A) The vRLGS image with the extra spot filtering off. Extra spots are indicated with red Xs. Spot A is a spot of interest for which the presence of extra spots makes the selection of the best candidate sequence difficult. B) The same vRLGS image with the extra spot filtering on. Faded red Xs indicate the positions the extra spot occupied in A), but are not seen with the filtering on. The candidate sequence for spot of interest A becomes immediately obvious. C) Example of PCR candidate sequence confirmation. DNA was eluted from the spots indicate as template DNA for PCR using primers derived from the candidate sequence for the spots of interest. In the last two lanes, primers for the candidate sequence for spot 68 were used to amplify DNA eluted from spot 62 and spot 68. Only the when the DNA eluted from spot 68 was used as template was a band observed. This is confirmation that the sequence predicted by the vRLGS in A) and B) is correct for spot 68. The same is true for spots 36 and 56 (not shown in A) and B)), and 62.