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Quantification of rare allelic variants from pooled genomic DNA


We report a targeted, cost-effective method to quantify rare single-nucleotide polymorphisms from pooled human genomic DNA using second-generation sequencing. We pooled DNA from 1,111 individuals and targeted four genes to identify rare germline variants. Our base-calling algorithm, SNPSeeker, derived from large deviation theory, detected single-nucleotide polymorphisms present at frequencies below the raw error rate of the sequencing platform.

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Figure 1: Error modeling.
Figure 2: Allele frequency by sequencing versus genotyping.


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This work was supported in part by the US National Institutes of Health under the Ruth L. Kirschstein National Research Service Award T32 HD 007499 from the National Institute of Child Health and Human Development (T.E.D.), the National Heart, Lung and Blood Institute (RO1HL065174, RO1HL082747, F.S.C.), the Children's Discovery Institute Fellowship Award MC-F-2006-1 (T.E.D.), the Children's Discovery Institute grant MC-II-2006-1 (R.D.M.) and the Saigh Foundation (F.S.C., R.D.M. and T.E.D).

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Authors and Affiliations



T.E.D., R.D.M. and K.E.V. designed the experiments. T.E.D. performed the sequencing experiments. D.J.W. and S.W.R. performed the TaqMan assays. O.L.K. and J.A.B. punched the bloodspots from filter paper. F.L.M.V designed and wrote SNPSeeker. F.L.M.V. and R.D.M. analyzed data. J.C.F. and S.W.D. designed and executed the comparative genomic analysis. D.J.W., A.H. and F.S.C. provided reagents and advice. T.E.D., R.D.M., F.L.M.V. and K.E.V. wrote the manuscript.

Corresponding author

Correspondence to Robi D Mitra.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–5, Supplementary Methods, Supplementary Results (PDF 398 kb)

Supplementary Software

SNPSeeker README file and binaries for Linux 32-bit, 64-bit, and Mac OSX (ZIP 69 kb)

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Druley, T., Vallania, F., Wegner, D. et al. Quantification of rare allelic variants from pooled genomic DNA. Nat Methods 6, 263–265 (2009).

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