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Nucleic Acids Res. 2011 Mar;39(5):e28. doi: 10.1093/nar/gkq1249. Epub 2010 Dec 3.

A simple method using PyrosequencingTM to identify de novo SNPs in pooled DNA samples.

Author information

1
Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu 300, Taiwan. yslin@faculty.nctu.edu.tw

Abstract

A practical way to reduce the cost of surveying single-nucleotide polymorphism (SNP) in a large number of individuals is to measure the allele frequencies in pooled DNA samples. Pyrosequencing(TM) has been frequently used for this application because signals generated by this approach are proportional to the amount of DNA templates. The Pyrosequencing(TM) pyrogram is determined by the dispensing order of dNTPs, which is usually designed based on the known SNPs to avoid asynchronistic extensions of heterozygous sequences. Therefore, utilizing the pyrogram signals to identify de novo SNPs in DNA pools has never been undertook. Here, in this study we developed an algorithm to address this issue. With the sequence and pyrogram of the wild-type allele known in advance, we could use the pyrogram obtained from the pooled DNA sample to predict the sequence of the unknown mutant allele (de novo SNP) and estimate its allele frequency. Both computational simulation and experimental Pyrosequencing(TM) test results suggested that our method performs well. The web interface of our method is available at http://life.nctu.edu.tw/∼yslin/PSM/.

PMID:
21131285
PMCID:
PMC3061071
DOI:
10.1093/nar/gkq1249
[Indexed for MEDLINE]
Free PMC Article
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