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Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need...

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Detalles Bibliográficos
Autores principales: Callegaro, A., Spinelli, R., Beltrame, L., Bicciato, S., Caristina, L., Censuales, S., De Bellis, G., Battaglia, C.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440877/
https://www.ncbi.nlm.nih.gov/pubmed/16617143
http://dx.doi.org/10.1093/nar/gkl185
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author Callegaro, A.
Spinelli, R.
Beltrame, L.
Bicciato, S.
Caristina, L.
Censuales, S.
De Bellis, G.
Battaglia, C.
author_facet Callegaro, A.
Spinelli, R.
Beltrame, L.
Bicciato, S.
Caristina, L.
Censuales, S.
De Bellis, G.
Battaglia, C.
author_sort Callegaro, A.
collection PubMed
description Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.
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spelling pubmed-14408772006-05-04 Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data Callegaro, A. Spinelli, R. Beltrame, L. Bicciato, S. Caristina, L. Censuales, S. De Bellis, G. Battaglia, C. Nucleic Acids Res Methods Online Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing. Oxford University Press 2006 2006-04-14 /pmc/articles/PMC1440877/ /pubmed/16617143 http://dx.doi.org/10.1093/nar/gkl185 Text en © The Author 2006. Published by Oxford University Press. All rights reserved
spellingShingle Methods Online
Callegaro, A.
Spinelli, R.
Beltrame, L.
Bicciato, S.
Caristina, L.
Censuales, S.
De Bellis, G.
Battaglia, C.
Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title_full Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title_fullStr Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title_full_unstemmed Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title_short Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data
title_sort algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of taqman real-time data
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1440877/
https://www.ncbi.nlm.nih.gov/pubmed/16617143
http://dx.doi.org/10.1093/nar/gkl185
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