Mixed Sequence Reader: A Program for Analyzing DNA Sequences with Heterozygous Base Calling
Figure 5
Experimental confirmation of HSPDP3 and DEFB4 copy numbers identified by Mixed Sequence Reader. (a) The primers used to amplify the DEF4B and HSPDP3 genes in the human genome. The LRi ratio between DEFB4 and HSPDP3 was calculated for each chromatography trace. The copy number of HSPDP3 is constant in the human genome (). (b) The heterozygous chromatography trace of PCR products comprising both HSPDP3 and DEFB4. The median DEFB4/HSPDP3 ratio was 1.63, which was estimated by MSR program. (c) The PCR product from (b) was cloned, and at least twenty colonies were picked for DNA sequencing to calculate the ratio between DEFB4 and HSPDP3. The observed ratio was 1.66, compatible with that derived from MSR. (d) Comparison of median LRi values from the forward and reverse primers. Using the k-means algorithm, pairs of median LRi values were clustered into 5 groups corresponding to different copy numbers (2, 3, 4, 5, and 6). Five samples (marked with circles) were cloned into the T and A vectors to validate the DEFB4 copy number.
