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Identification of genetic variants using bar-coded multiplexed sequencing


We developed a generalized framework for multiplexed resequencing of targeted human genome regions on the Illumina Genome Analyzer using degenerate indexed DNA bar codes ligated to fragmented DNA before sequencing. Using this method, we simultaneously sequenced the DNA of multiple HapMap individuals at several Encyclopedia of DNA Elements (ENCODE) regions. We then evaluated the use of Bayes factors for discovering and genotyping polymorphisms. For polymorphisms that were either previously identified within the Single Nucleotide Polymorphism database (dbSNP) or visually evident upon re-inspection of archived ENCODE traces, we observed a false positive rate of 11.3% using strict thresholds for predicting variants and 69.6% for lax thresholds. Conversely, false negative rates were 10.8–90.8%, with false negatives at stricter cut-offs occurring at lower coverage (<10 aligned reads). These results suggest that >90% of genetic variants are discoverable using multiplexed sequencing provided sufficient coverage at the polymorphic base.

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Figure 1
Figure 2: Comparison of index performance.
Figure 3: Relationship between mean and local coverage.
Figure 4: Discovery of variant bases by simultaneous analysis of all individuals.
Figure 5: Relationship between base-level coverage and Bayes factor for polymorphism discovery and variant genotyping.

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We acknowledge funding from the state of Arizona, US National Heart Lung and Blood Institute (U01 HL086528), the Stardust foundation, Science Foundation Arizona, and National Institute for Neurological Disorders and Strokes (R01 N5059873).

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D.W.C., J.V.P., M.J.H., G.N. and D.A.S. contributed to initial experimental design. S.S., A.S., M.R., J.J.C., T.L. and T.L.P. contributed to development and execution of exact experimental protocols. J.V.P., D.W.C. and N.H. contributed to the development of bioinformatics and analysis pipelines.

Corresponding author

Correspondence to David W Craig.

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Competing interests

G.N. is an employee of Illumina.

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Supplementary Figures 1–2, Supplementary Tables 1–5, Supplementary Methods (PDF 434 kb)

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Craig, D., Pearson, J., Szelinger, S. et al. Identification of genetic variants using bar-coded multiplexed sequencing. Nat Methods 5, 887–893 (2008).

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