High-throughput genotyping on archived dried blood spot samples.
Author(s): Hollegaard MV, Grove J, Thorsen P, Nørgaard-Pedersen B, Hougaard DM
Publication: Genet Test Mol Biomarkers, 2009, Vol. 13, Page 173-9
PubMed ID: 19371215 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of method of whole genome amplification (WGA), storage duration, and filter paper used for collection on SNP analysis of dried blood specimens (DBS).
Conclusion of Paper
GPlex was better suited than the AmpliQ kit for WGA of 19 year old DBS based on SNP assay call frequencies. In specimens stored for longer than 21 years, a significantly lower amount of genomic DNA (gDNA) was obtained and used for the WGA reaction, but a higher concentration of amplified DNA was obtained. Use of the amplified DNA obtained from these specimens resulted in genotype calls that were of lower quality and reliability when compared to specimens stored for 19-21 years indicating that the increased DNA was from non-specific amplification. Furthermore, the 47/644 specimens that failed genotyping were all stored longer than 21 years. Specimens collected on S&S903 filter paper yielded more gDNA when compared to specimens collected on S&S2992 filter paper but no effect of filter paper on WGA concentration was found. However, specimens collected on S&S2992 filter paper gave the highest quality and most reliable genotype calls.
Studies
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Study Purpose
The purpose of this study was to determine the optimum method of WGA from 19 year old DBS. In addition, the protocol was used to compare new DBS with old ones.
Summary of Findings:
GPlex was better suited than the AmpliQ kit for WGA of 19 year old DBS based on SNP assay call frequencies. The average gDNA extracted from new DBS was 272 ng versus 19 year old DBS which yielded an average of 70 ng. However, the authors note that newly spotted specimens contained 8 times more blood than standard DBS.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Not specified
Platform:
Analyte Technology Platform DNA Whole genome amplification DNA SNP assay DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Whole genome amplification Specific Nucleic acid amplification AmpliQ Genomic Amplifier kit
GPlex
Storage Storage duration 0 y
19 y
Storage Storage temperature -24 degrees C
-
Study Purpose
The purpose of this study was to determine the effects of storage duration and filter paper used to collect DBS on gDNA yield, WGA, and subsequent SNP analysis. Specimens from two biobanks were used. One biobank held specimens collected from newborns without EDTA at -24 degrees C for 19 to 27 years. A different biobank held specimens from pregnant women collected with EDTA at -20 degrees for 9 to 11 years.
Summary of Findings:
Genotyping results were obtained through SNP analysis in 92.7% of 644 specimens. All 47 specimens that failed genotyping were newborn specimens without EDTA and were stored between 21 and 27 years at -24 degrees C. In specimens stored for longer than 21 years, a significantly lower amount of gDNA was obtained and used for the WGA reaction, but a higher concentration of amplified DNA was obtained. Use of the amplified DNA obtained from these specimens resulted in genotype calls that were of lower quality and reliability indicating non-specific amplification when compared to specimens stored for 19-21 years. Specimens stored on S&S903 filter paper yielded more gDNA than specimens stored on S&S2992 filter paper, but no effect of filter paper on WGA concentration was found. However, specimens collected on S&S2992 filter paper gave the highest quality and most reliable genotype calls.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Not specified
- Pregnant
Platform:
Analyte Technology Platform DNA Whole genome amplification DNA SNP assay DNA Fluorometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Type of collection container/solution S&S2992 filter paper
S&S903 filter paper
Storage Storage duration 9-11 y
19-27 y
Storage Storage temperature -20 degrees C
-24 degrees C
Preaquisition Patient age Newborn
Pregnant adult
Biospecimen Acquisition Anticoagulant EDTA
None