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DNA specimens from 23 previously studied HNPCC patients were analyzed by DHPLC, and all mutations were correctly identified and confirmed by sequence analysis.
Although there is some subjectivity in this analysis, we found that ∼80% of SWISS-PROT and ∼64% of PMD mutations were correctly classified as activating, which typically required mention of increased enzymatic activity or substrate binding.
Using the Fluidigm custom panel, 20 of the 22 known mutations were correctly identified.
All mutations were correctly detected by the PCR assay in the 22 samples tested.
If all mutations were correctly identified in both validations, the measured sensitivity would be 100% in both cases.
Only 26, 46 and 38% of mutations were correctly diagnosed to be neutral by Condel, PolyPhen-2 and SIFT, respectively (Fig. 4A).
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The read depth has much less effect on the estimation, particularly if the read depth is >30; even for read depth as low as 10, the loss of precision due to low read depth is not nearly as striking as that due to reducing the number of mutations (assuming that the mutations are correctly identified as mutated, which is problematic with only 10× coverage).
At a B-SIFT score cutoff of 0.5, we observe a 9% sensitivity towards identifying activating mutations but a 99% specificity, suggesting that we are able to identify only a subset of activating mutations but the majority of mutations are correctly classified as non-activating.
The insert was sequenced to verify that the intended mutation was correctly constructed.
Each mutation was correctly identified in each exon giving 100% sensitivity for HRM within this sample set.
Conversely, mutation was correctly incorporated as the source of new alleles in the population (3 points) significantly more frequently in students' models than in short answers (Fisher's exact test, p < 0.0001).
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com