Evaluation of a New Pooling Strategy Based on Leukocyte Count for Rapid Quantification of Allele Frequencies
Heidi Rossmann,Elena Büchler,Jürgen J. Wenzel,Carolin Neukirch,Jean-Baptist du Prel,Karl J. Lackner +5 more
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TLDR
DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples and the leukocytes approach is much less tedious, quicker, and less expensive.Abstract:
Background: Allele frequencies of single-nucleotide polymorphisms (SNPs) can be quantified from DNA pools. The conventional preparation of DNA pools requires DNA isolation and quantification for each blood sample. We hypothesized that pooling of whole blood samples according to their leukocyte count, which determines DNA content, would be as reliable as the conventional pooling method but much less tedious to perform.
Methods: We collected 100 whole blood samples and measured the leukocyte count. Samples were frozen until further use. After thawing, pools were generated by combining aliquots containing an equal number of leukocytes. In parallel, DNA was extracted from another aliquot, DNA concentration was measured, and DNA concentration-based pools were assembled. All original samples were genotyped directly using 4 different SNP assays to obtain the exact allele frequencies in the pool. In addition, samples of known genotypes were mixed according to the DNA concentration or the leukocyte count to generate artificial samples of known allele frequencies. We analyzed pools and mixes in triplicate by pyrosequencing and calculated allelic frequencies.
Results: Leukocyte and DNA pooling provided equally accurate and precise SNP frequencies comparable to published data.
Conclusion: DNA and leukocyte pooling are both suitable strategies to determine allele frequencies in frozen samples. The leukocyte pooling approach is much less tedious, quicker, and less expensive. It should be always considered if leukocyte counts are available.read more
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Identification and Prevention of Genotyping Errors Caused by G-Quadruplex– and i-Motif–Like Sequences
Jürgen J. Wenzel,Heidi Rossmann,Christian Fottner,Stefan Neuwirth,Carolin Neukirch,Peter Lohse,Julia K. Bickmann,Timo Minnemann,Thomas J. Musholt,Brigitte Schneider-Rätzke,Matthias M. Weber,Karl J. Lackner +11 more
TL;DR: It is shown that G- quadruplex- or i-motif-like sequences can reproducibly cause ADO, and PCR products should be checked for G-quadruplex and i-Motifs to avoid the formation of ADO-causing secondary structures.
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Phenotypic variability and risk of malignancy in SDHC-linked paragangliomas: lessons from three unrelated cases with an identical germline mutation (p.Arg133*).
Julia K. Bickmann,Stefanie Sollfrank,Arno Schad,Thomas J. Musholt,Erik Springer,Matthias Miederer,Oliver Bartsch,Konstantinos Papaspyrou,Dimitrios Koutsimpelas,Wolf J. Mann,Matthias M. Weber,Karl J. Lackner,Heidi Rossmann,Christian Fottner +13 more
TL;DR: The present case shows that SDHC germline mutations can have highly variable phenotypes and may cause malignant PGL, although malignancy is probably rare.
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Novel GATA1 Variant Causing a Bleeding Phenotype Associated with Combined Platelet α-/δ-Storage Pool Deficiency and Mild Dyserythropoiesis Modified by a SLC4A1 Variant
Kerstin Jurk,Anke Adenaeuer,Stefanie Sollfrank,Kathrin Gross,Friederike Häuser,Andreas Czwalinna,J. Erkel,Nele Fritsch,D Marandiuc,Martin Schaller,Karl J. Lackner,Heidi Rossmann,Frauke Bergmann +12 more
TL;DR: Flow cytometry and electron microscopy analysis supported a combined α-/δ (AN-subtype)-storage pool deficiency as cause for impaired agonist-induced platelet aggregation and granule exocytosis and the absence of BCAM in the index and its low expression in the daughter confirmed a less obvious effect of defective GATA1 also on erythrocytes.
References
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DNA Pooling: a tool for large-scale association studies
TL;DR: Recent developments in quantitative genotyping assays and in the design and analysis of pooling studies are discussed.
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Determination of Single-Nucleotide Polymorphisms by Real-time Pyrophosphate DNA Sequencing
TL;DR: This work addresses the issue of SNP genotype determination by investigating variations within the Renin-Angiotensin-Aldosterone System using pyrosequencing, a real-time pyrophosphate detection technology and provides extensive flexibility in regard to the positioning of sequencing primers.
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Rapid SNP allele frequency determination in genomic DNA pools by pyrosequencing.
TL;DR: This work has developed an accurate and reproducible protocol for allele frequency determination using Pyrosequencing technology in large genomic DNA pools (374 individuals), which allows the rapid determination of allelefrequency differences in case/control groups for association studies and susceptibility gene discovery in complex diseases.
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Quantitative technologies for allele frequency estimation of SNPs in DNA pools
TL;DR: It is found that the influence of the error variance attributed to pool construction on quantification accuracy is insignificant and is SNP dependent, while the methods can all serve for quantification of allele frequency in DNA pools with reasonable accuracy.
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Pyrosequencing™‐based SNP allele frequency estimation in DNA pools
TL;DR: Pyrosequencing™ can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios with accuracy and precision values in ranges comparable with those of other SNP typing techniques.