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. 2010 May;7(5):336-7.
doi: 10.1038/nmeth0510-336.

Intensity Normalization Improves Color Calling in SOLiD Sequencing

Free PMC article

Intensity Normalization Improves Color Calling in SOLiD Sequencing

Hao Wu et al. Nat Methods. .
Free PMC article

Abstract

ABI’s SOLiD system is a commonly used massively parallel DNA sequencing platform for applications including genotyping and structural variation analysis to transcriptome quantification and reconstruction. Like other sequencing technologies, it measures fluorescence intensities from dye-labeled molecules to determine the sequence of DNA fragments. Ultimately, sequences are determined by complicated statistical manipulations of noisy intensity measurements but systematic biases may mislead downstream analysis. A number of proposed methods improve base-calling and quality metrics for other sequencing technologies- and we now present Rsolid, software implementing an intensity normalization strategy for the SOLiD platform that substantially improves yield and accuracy at small computational costs (7% increase in total matches, 13% in perfect matches, 5% reduced error rate, and substantial reduction in false-positive SNP calls).

Figures

Figure 1
Figure 1. Effect of normalization on color proportions and SNP calling
(a) Color proportions in sample of E. coli genomic DNA on each sequencing cycle. Color calls as reported by the SOLiD 2 system (left panel), and after normalization by Rsolid (right panel), (b) Number of false-positive SNPs called in E. coli as coverage increases. Observe that after normalization fewer SNPs are called even at high coverage (30 M reads corresponds to ~100x coverage).

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