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Open Access Highly Accessed Research article

The effect of strand bias in Illumina short-read sequencing data

Yan Guo1, Jiang Li1, Chung-I Li1, Jirong Long2, David C Samuels3 and Yu Shyr1*

Author Affiliations

1 Vanderbilt Ingram Cancer Center, Center for Quantitative Sciences, Nashville, TN, USA

2 Vanderbilt Epidemiology Center, Vanderbilt University School of Medicine, Nashville, TN, USA

3 Center for Human Genetics Research, Vanderbilt University, Nashville, TN, USA

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BMC Genomics 2012, 13:666  doi:10.1186/1471-2164-13-666

Published: 24 November 2012

Abstract

Background

When using Illumina high throughput short read data, sometimes the genotype inferred from the positive strand and negative strand are significantly different, with one homozygous and the other heterozygous. This phenomenon is known as strand bias. In this study, we used Illumina short-read sequencing data to evaluate the effect of strand bias on genotyping quality, and to explore the possible causes of strand bias.

Result

We collected 22 breast cancer samples from 22 patients and sequenced their exome using the Illumina GAIIx machine. By comparing the consistency between the genotypes inferred from this sequencing data with the genotypes inferred from SNP chip data, we found that, when using sequencing data, SNPs with extreme strand bias did not have significantly lower consistency rates compared to SNPs with low or no strand bias. However, this result may be limited by the small subset of SNPs present in both the exome sequencing and the SNP chip data. We further compared the transition and transversion ratio and the number of novel non-synonymous SNPs between the SNPs with low or no strand bias and those with extreme strand bias, and found that SNPs with low or no strand bias have better overall quality. We also discovered that the strand bias occurs randomly at genomic positions across these samples, and observed no consistent pattern of strand bias location across samples. By comparing results from two different aligners, BWA and Bowtie, we found very consistent strand bias patterns. Thus strand bias is unlikely to be caused by alignment artifacts. We successfully replicated our results using two additional independent datasets with different capturing methods and Illumina sequencers.

Conclusion

Extreme strand bias indicates a potential high false-positive rate for SNPs.

Keywords:
Next Generation Sequencing; Strand Bias; Illumina; Short Read; SNP quality control