Limits...
Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping.

Bucasas KL, Pandya GA, Pradhan S, Fleischmann RD, Peterson SN, Belmont JW - BMC Genet. (2009)

Bottom Line: Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results.Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold.We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA. lacuesta@bcm.edu

ABSTRACT

Background: Whole genome amplification (WGA) offers new possibilities for genome-wide association studies where limited DNA samples have been collected. This study provides a realistic and high-precision assessment of WGA DNA genotyping performance from 20-year old archived serum samples using the Affymetrix Genome-Wide Human SNP Array 6.0 (SNP6.0) platform.

Results: Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold.

Conclusions: We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.

Show MeSH
Performance analysis of technical replicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2803178&req=5

Figure 3: Performance analysis of technical replicate samples.

Mentions: We further evaluated the quality of SNP 6.0 genotyping performance on WGA serum DNA samples by measuring the repeatability of calls between technical replicates as shown in Figure 3 (see Additional File 3). Reports for concordance were restricted to SNP loci with complete genotype calls on both technical replicates of a given individual. Genotype data between technical replicates gave modest percent concordances, ranging from 77.3 to 99.9 (mean +/- SD, 95.2 +/- 4.6) over approximately 340,000 SNP loci. We then determined the percent contribution of allele switch (AA ←→ BB) or heterozygote dropout (AB → AA or BB) to the observed global discordance as shown in Figure 3 (see Additional File 4). In all technical samples, discordance occurred largely due to heterozygote dropout, with an average of 85.9%. Allele switch was less common and contributed to 14.1% of the global discordance.


Assessing the utility of whole-genome amplified serum DNA for array-based high throughput genotyping.

Bucasas KL, Pandya GA, Pradhan S, Fleischmann RD, Peterson SN, Belmont JW - BMC Genet. (2009)

Performance analysis of technical replicate samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803178&req=5

Figure 3: Performance analysis of technical replicate samples.
Mentions: We further evaluated the quality of SNP 6.0 genotyping performance on WGA serum DNA samples by measuring the repeatability of calls between technical replicates as shown in Figure 3 (see Additional File 3). Reports for concordance were restricted to SNP loci with complete genotype calls on both technical replicates of a given individual. Genotype data between technical replicates gave modest percent concordances, ranging from 77.3 to 99.9 (mean +/- SD, 95.2 +/- 4.6) over approximately 340,000 SNP loci. We then determined the percent contribution of allele switch (AA ←→ BB) or heterozygote dropout (AB → AA or BB) to the observed global discordance as shown in Figure 3 (see Additional File 4). In all technical samples, discordance occurred largely due to heterozygote dropout, with an average of 85.9%. Allele switch was less common and contributed to 14.1% of the global discordance.

Bottom Line: Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results.Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold.We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA. lacuesta@bcm.edu

ABSTRACT

Background: Whole genome amplification (WGA) offers new possibilities for genome-wide association studies where limited DNA samples have been collected. This study provides a realistic and high-precision assessment of WGA DNA genotyping performance from 20-year old archived serum samples using the Affymetrix Genome-Wide Human SNP Array 6.0 (SNP6.0) platform.

Results: Whole-genome amplified (WGA) DNA samples from 45 archived serum replicates and 5 fresh sera paired with non-amplified genomic DNA were genotyped in duplicate. All genotyped samples passed the imposed QC thresholds for quantity and quality. In general, WGA serum DNA samples produced low call rates (45.00 +/- 2.69%), although reproducibility for successfully called markers was favorable (concordance = 95.61 +/- 4.39%). Heterozygote dropouts explained the majority (>85% in technical replicates, 50% in paired genomic/serum samples) of discordant results. Genotyping performance on WGA serum DNA samples was improved by implementation of Corrected Robust Linear Model with Maximum Likelihood Classification (CRLMM) algorithm but at the loss of many samples which failed to pass its quality threshold. Poor genotype clustering was evident in the samples that failed the CRLMM confidence threshold.

Conclusions: We conclude that while it is possible to extract genomic DNA and subsequently perform whole-genome amplification from archived serum samples, WGA serum DNA did not perform well and appeared unsuitable for high-resolution genotyping on these arrays.

Show MeSH