Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and Next-Generation Sequencing
Figure 2
Variant validation within RET intron 9 by high-resolution melting analysis. The fluorescence difference plot (fluorescence difference versus temperature) of the melting curve data is shown in each panel. (a) RET intron 9. The black lines are samples of homozygous wild-type sequence. Data from four samples with unique variants within intron 9 are shown: heterozygous at amplicon position 156 (green trace, c.1760−158C>G), heterozygous at 174 (red trace, c.1760−140C>G), heterozygous at 117 (pink trace, c.1760−197G>T), and a sample heterozygous at both positions 117 and 174 (light blue). (b) Intron 9 with wild-type DNA spiked into the PCR reaction to help differentiate homozygous variants. One sample with a homozygous variant at position 174 with (“+spike”) and without wild-type DNA spiked in is shown (blue traces) compared to a 174 heterozygous (red trace) and wild-type sample (black trace).
