Effects of DNA mass on multiple displacement whole genome amplification and genotyping performance.
BACKGROUND
Whole genome amplification (WGA) promises to eliminate practical molecular genetic analysis limitations associated with genomic DNA (gDNA) quantity. We evaluated the performance of multiple displacement amplification (MDA) WGA using gDNA extracted from lymphoblastoid cell lines (N = 27) with a range of starting gDNA input of 1-200 ng into the WGA reaction. Yield and composition analysis of whole genome amplified DNA (wgaDNA) was performed using three DNA quantification methods (OD, PicoGreen and RT-PCR). Two panels of N = 15 STR (using the AmpFlSTR Identifiler panel) and N = 49 SNP (TaqMan) genotyping assays were performed on each gDNA and wgaDNA sample in duplicate. gDNA and wgaDNA masses of 1, 4 and 20 ng were used in the SNP assays to evaluate the effects of DNA mass on SNP genotyping assay performance. A total of N = 6,880 STR and N = 56,448 SNP genotype attempts provided adequate power to detect differences in STR and SNP genotyping performance between gDNA and wgaDNA, and among wgaDNA produced from a range of gDNA templates inputs.


RESULTS
The proportion of double-stranded wgaDNA and human-specific PCR amplifiable wgaDNA increased with increased gDNA input into the WGA reaction. Increased amounts of gDNA input into the WGA reaction improved wgaDNA genotyping performance. Genotype completion or genotype concordance rates of wgaDNA produced from all gDNA input levels were observed to be reduced compared to gDNA, although the reduction was not always statistically significant. Reduced wgaDNA genotyping performance was primarily due to the increased variance of allelic amplification, resulting in loss of heterozygosity or increased undetermined genotypes. MDA WGA produces wgaDNA from no template control samples; such samples exhibited substantial false-positive genotyping rates.


CONCLUSIONS
The amount of gDNA input into the MDA WGA reaction is a critical determinant of genotyping performance of wgaDNA. At least 10 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain wgaDNA TaqMan SNP assay genotyping performance equivalent to that of gDNA. Over 100 ng of lymphoblastoid gDNA input into MDA WGA is required to obtain optimal STR genotyping performance using the AmpFlSTR Identifiler panel from wgaDNA equivalent to that of gDNA.
Bergen
Andrew W
AW
Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. bergena@mail.nih.gov
Qi
Ying
Y
Haque
Kashif A
KA
Welch
Robert A
RA
Chanock
Stephen J
SJ
D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D005189:False Positive Reactions; D005821:Genetic Techniques; D016678:Genome; D005838:Genotype; D006801:Humans; D019656:Loss of Heterozygosity; D008214:Lymphocytes / Q000378:metabolism; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D009930:Organic Chemicals / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D011786:Quality Control; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA