Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms.
Highly accurate and high-throughput SNP genotyping platforms are increasingly popular but the performance of suboptimal DNA samples remains unclear. The aim of our study was to determine the best platform, amplification technique, and loading concentration to maximize genotype accuracy and call rate using degraded samples. We amplified high-molecular weight genomic DNA samples recently extracted from whole blood and degraded DNA samples extracted from 50-year-old patient sera. Two whole-genome amplification (WGA) methodologies were used: an isothermal multiple displacement amplification method (MDA) and a fragmentation-PCR-based method (GenomePlex [GPLEX]; Sigma-Aldrich, St. Louis, MO). Duplicate runs were performed on genome-wide dense SNP arrays (Nsp-Mendel; Affymetrix) and custom SNP platforms based on molecular inversion probes (Targeted Genotyping [TG]; Affymetrix) and BeadArray technology (Golden Gate [GG]; Illumina). Miscalls and no-calls on Mendel arrays were correlated with each other, with confidence scores from the Bayesian calling algorithm, and with average probe intensity. Degraded DNA amplified with MDA gave low call rates and concordance across all platforms at standard loading concentrations. The call rate with MDA on GG was improved when a 5 x concentration of amplified DNA was used. The GPLEX amplification gave high call rate and concordance for degraded DNA at standard and higher loading concentrations on both TG and GG platforms. Based on these analyses, after standard filtering for SNP and sample performance, we were able to achieve a mean call rate of 99.7% and concordance 99.7% using degraded samples amplified by GPLEX on GG technology at 2 x loading concentration. These findings may be useful for investigators planning case-control association studies with patient samples of suboptimal quality.
Mead
Simon
S
Medical Research Council (MRC) Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, London, United Kingdom.
Poulter
Mark
M
Beck
John
J
Uphill
James
J
Jones
Chris
C
Ang
Cheng Eng
CE
Mein
Charles A
CA
Collinge
John
J
D004247:DNA / Q000378:metabolism*; D005821:Genetic Techniques; D015894:Genome, Human; D006801:Humans; D007729:Kuru / Q000235:genetics; D008875:Middle Aged; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020641:Polymorphism, Single Nucleotide
D004247:DNA