Genotyping Support—Troubleshooting

If gDNA preparation is suspected to contain inhibitors, the following cleanup procedure can be used:

1. Add 0.5 volumes of 7.5 M NH4OAc, 2.5 volumes of absolute ethanol (stored at –20 °C), to gDNA.
2. Vortex and incubate at –20 °C for 1 hr.
3. Centrifuge at 12,000 x g in a microcentrifuge at room temperature for 20 min.
4. Remove supernatant and wash pellet with 80% ethanol.
5. Centrifuge at 12,000 x g at room temperature for 5 min.
6. Remove the 80% ethanol and repeat the 80% ethanol wash one more time.
7. Resuspend the pellet in reduced EDTA TE Buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA).

The Mendelian consistency rate of the validated SNPs on the Axiom™ Genome-Wide BOS 1 Array Plate is 99.96 percent among different experiments.

The Mendelian consistency rate of the validated SNPs on the Axiom™ Genome-Wide BOS 1 Array Plate is 99.96 percent among different experiments.

The reproducibility for the validated SNPs on the screening arrays is 99.94 percent.

The performance of the array was measured across 20 breeds, and the average sample call rate is 99.62 percent.

We have performance metrics for using the Mouse Diversity Genotyping Array with classic inbred laboratory strains and F1 crosses between these inbred strains, but we do not currently offer comprehensive metrics for wild-derived strains.

Table 2 of the supplement to the publication “A customized and versatile high-density genotyping array for the mouse” (Yang H., et al., Nature Methods, 2009), shows performance (call, heterozygosity, and concordance rates) of the array for six wild-derived strains: WSB/EiJ, PWD/EiJ, MOLF/EiJ, CAST/EiJ, SPRET/EiJ, and PANCEVO/EiJ, representing M. m. domesticus, M. M. musculus, M. m. castaneus, M. m. molosinnus subspecies, and M. spretus and M. spicilegus species, respectively. The array performance was highest for wild-derived strains from M. m. domesticus subspecies, followed by strains from other Mus subspecies, which reflects the degree of genetic divergence from the reference strain used C57BL/6J. The least divergent strain, WSB/EiJ, had the highest call and concordance rates (98.041, 0.992) and lowest heterozygosity rate (2.445), while the most divergent strain, SPRET/EiJ, had the lowest call and concordance rates (92.386, 0.986) and highest heterozygosity rate (14.797).

In a study by The Jackson Laboratory comparing two inbred strains, 99.7 percent of the called genotypes were correct. Among the 114,625 polymorphisms detected, 112,759 SNPs had genotypes for these strains in the Single Nucleotide Polymorphism database (dbSNP). 99.7% (112,452) of the SNPs had the same genotypes between the Mouse Diversity Genotyping Array analysis results and the dbSNP. Among the 307 discordant SNPs, 301 were genotyped by Perlegen and the other five were computed genotypes from the Sanger Institute.