While STR allele calls may be made in an automated fashion with either Genotyper® or STaR Call™, the resulting genotype information needs to be manually examined by experienced analysts. In Chapter 17, we will discuss several expert systems (i.e., computer programs) that are under development to perform automated STR data review. Data analysis and review is essential for confirming STR results prior to making reports.
Software algorithms follow set parameters and criteria and hence can never be as effective at making difficult calls as a trained examiner. Strict guidelines for data interpretation should be in place to avoid problems with individual bias when the data is reviewed. However, there is always enough variation between data sets that not every situation can be covered by a pre-determined rule.
Laboratories typically have two independent reads of the data by different operators. The genotypes must agree with each other before results will be reported or passed on for uploading to a DNA database. Likewise, a match between two samples is only reported if the two DNA profiles display the same pattern.
There are a number of issues that are important to obtaining accurate genotype results. Some issues are biology related and some are technology related. For example, the amount of stutter or incomplete 3' nucleotide addition present are biology issues related to the amount of DNA template used in the PCR amplification. On the other hand, 'pull-up' artifacts and threshold issues result from the fluorescent technology and software used for genotyping the samples.
Three parts of the genotyping process illustrated in Figure 15.1 are crucial to the success of genotyping samples. These include the matrix file, the internal size standard, and the allelic ladder sample.
The matrix file (termed a spectral calibration for ABI 3100 users) is critical for proper color separation in an electropherogram. If the observed peaks are not associated with the proper dye label, then the sample genotype cannot be correctly determined. Matrix files are established by running samples that contain each of the dyes individually. The results of the individual dye runs are combined to form a mathematical matrix that is used to subtract the contribution of other colors in the overlapping spectra (see Chapter 13). A matrix is most accurate under consistent environmental conditions. Thus, if the electro-phoresis buffer is changed, a new matrix should be established in order to obtain the most accurate color deconvolution between the different dyes.
The internal size standard is necessary for properly sizing of DNA fragment peaks detected in an electropherogram. If any of the peaks in the size standard are below the peak detection threshold established in the data collection and analysis software, then the sizing algorithms will not work properly and STR alleles may be sized incorrectly. An analyst should check to make sure that the internal size standard peaks were all detected properly before proceeding to genotype the STR alleles in a sample.
The allelic ladder is the standard to which STR alleles are compared to obtain the sample genotype. The alleles in an allelic ladder need to be resolved from one another and above the peak detection threshold of the data collection and analysis software in order to correctly call STR alleles in unknown samples. The sizes obtained for each allele in the allelic ladder are used to make the final genotype determination in the unknown samples. Therefore, they must be determined correctly.
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