Figure 2: Repeat content and GC percentage across the 24 kb target region described in the text.

The figure is based on analysis of each 25 bp sequence (26 bp sliding window) of the plus strand. For all subfigures, red lines show the number of thermoalignments with an off-target T_m within 10 °C of the corresponding on-target T_m. Yellow lines (orange when overlapping with red) show the number of thermoalignments between a given primer and off-target sites with ≥70 percent identity (pid). Blue lines show the percent GC content. The search for off-target sites was based on BLASTn settings used in this study for priming specificity evaluation (see Methods), which had a maximum of 20 potential sites per pseudomolecule or a total of 260 possible sites. (a) Cumulative distribution of the number of repeats and percent GC content. (b) Genomic distribution of repeat content and GC percentage. The pseudomolecule coordinate of the 5′-nucleotide of each 25 bp sequence was used to position the plotted data. Black horizontal bars on the x-axis show the two genes in this region [left: GRMZM2G031364; right: GRMZM2G031239]. Among 25-mers in the region ≈73% would be predicted to have a misprime T_m within 10 °C of the primer T_m. (c) The CIRCOS plot extends from a single primer in the region with the greatest number (n = 215) of predicted mispriming sites across the genome. Red lines of the CIRCOS plot connect the predicted mispriming sites on the pseudomolecules for chromosomes 1 to 10, the mitochondria (Mt), the plastid (Pt) and unmapped sequences (unkn).