Experimental Workflow:
DNA Genotyping from Human FFPE Samples

Step 4. Analyze data and call alleles

In this final step, a scatter plot is generated from the normalized allelic discrimination data and alleles are called to determine the genotype of each sample. The inclusion of samples with <5% functional template may interfere with the AutoCall feature of the Sequence Detection Systems (SDS) Software. If this occurs, the alleles can be called manually. The allelic discrimination plot of a typical experiment contains four separate, distinct clusters which represent the no-template controls and three possible genotypes.

>> Recommendation: Collect data by post-plate read on the 7900HT Fast Real-Time PCR System, and use the AutoCall feature of the SDS software to accurately and reproducibly call alleles.

Case Study

After amplification, the reactions were evaluated via SDS 2.2.1 software using the post-plate read and AutoCalling (quality value threshold: 95) features.

The reproducibility of our recommended SNP genotyping procedure is demonstrated in Figure 3, which shows typical replicate allelic discrimination plots.

The reliability of this procedure is indicated by the results for 10 different TaqMan SNP genotyping assays, which are summarized in Figure 4. For samples containing at least 5% functional template, there is high correlation between observed and expected genotype frequencies. When samples containing less than 5% functional template are included in the analysis, the percent of samples that can be genotyped decreases. Taken together, these data suggest that to ensure that the AutoCall assigns a genotype to a sample, it is important to use only DNA samples that contain >5% functional template as determined by RNase P detection. Otherwise, all samples should be assayed and alleles called manually if the AutoCall feature is unable do so.

Figure 3. Achieve Highly Reproducible and Reliable Data from TaqMan® SNP Genotyping Assay. 1 ng, as quantified by RNase P detection, of each human FFPE DNA sample (n=105) was amplified in replicate for 60 cycles followed by AutoCalling using SDS 2.2.1 Software. Gene Symbol: GABRA2, Assay ID: C___8263011_10

				Observed Genotype Frequencies				Expected Genotype Frequencies
AB Assay ID	Amplicon Size (bp)	Assay Gene Symbol	dbSNP rs#	A1/A1	A2/A2	A1/A2	Total Samples	A1/A1	A2/A2	A1/A2	% Geno-typed	% Geno-typed*
C___1256256_1_	143	SASH1	rs2272998	11	27	35	73	11.1	27.1	34.7	99	81
C___8263011_10	132	GABRA2	rs279844	18	22	41	81	18.3	22.3	40.4	99	89
C___2515223_10	114	SYNE1	rs214955	21	18	43	82	22	19	40.9	99	92
C____411273_10	105	THSD2	rs2503107	24	14	43	81	25.6	15.6	39.9	100	87
C___1880371_10	104	RCHY1	rs13134862	13	31	37	81	12.3	30.3	38.5	100	97
C___7538108_10	81	SORBS1	rs1410059	21	22	37	80	19.5	20.5	40	98	92
C___9371416_10	72	HIVEP1	rs132184400	9	37	36	82	8.9	36.9	36.2	100	99
C___7459903_10	70	B4GALT6	rs985492	26	17	39	82	25.2	16.2	40.5	100	98
C___3254784_10	67	HSPA12A	rs740598	27	15	37	79	26.2	14.2	38.6	98	97
C___1619935_1_	65	PHGDHL1	rs1058083	13	29	39	81	13	29	38.9	99	96

* Includes samples with <5% functional template

Figure 4. Results Summary of TaqMan® SNP Genotyping Assays. Data was tabulated after removing all FFPE DNA samples with a ≤ 5% functional template. The “Expected Genotype Frequencies” (columns 9–11) are based on the gene frequencies of each polymorphism in the sample population. The observed genotype frequencies (columns 5–7) fit the expected genotype frequencies within a 95% confidence interval, using an appropriate chi-square test. Finally, the percent of samples genotyped is shown both before and after removal of the low quality samples (columns 12 and 13).