Fig. 2: Quantitative PCR confirms that intermediate read-depth profiles represent heterozygous deletions in L. infantum clones.

a HTZ_PI_2949 and HTZ_MT_3134 were selected as representatives of isolates for which read-depth drops to ca. 50% between 1.122 Mb and 1.135 Mb on chr31 (see Supplementary Fig. 4). DNA from monoclonal subcultures established from these two isolates was analyzed in qPCR targeting LinJ.31.2380 (within the chr31 deletion site) and LinJ.31.2330 (downstream of the chr31 deletion site). Differences in Ct values for LinJ.31.2330 between each HTZ sample and the NonDel reference (NonDel_MS_2666) were used to normalize a fold change estimate at LinJ.31.2380 based on the ∆∆Ct method by Livak and Schmittgen⁷⁵. Student’s t-test was applied to test whether fold change estimates obtained from n = 3 independent reactions differed significantly from the 1 : 1 ratio represented by the reference sample. Results were considered significant at *p < 0.05 and indicate that intermediate read-depth profiles represent abundant heterozygous deletions as opposed to mixtures of deletion-carrying and non-deletion-type cells within isolates. b Fold change was calculated the same way for monoclonal HTZ subcultures using the parental isolate as the reference. Results indicate that “unbalanced” heterozygotes also occur, e.g., subclone 2949 G1 appears to contain three chromosome copies with the chr31 deletion and one copy without.