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Open Access Highly Accessed Methodology article

Estimating DNA polymorphism from next generation sequencing data with high error rate by dual sequencing applications

Ziwen He1, Xinnian Li1, Shaoping Ling2, Yun-Xin Fu3, Eric Hungate4, Suhua Shi1* and Chung-I Wu124*

Author Affiliations

1 State Key Laboratory of Biocontrol and Guangdong Key Laboratory of Plant Resources, Sun Yat-sen University, 135 Xingang West Road, Guangzhou 510275, China

2 CAS Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, 1 Beichen West Road, Beijing 100101, China

3 Human Genetics Center, University of Texas School of Public Health, 1200 Herman Presser Drive, Houston, TX 77030, USA

4 Department of Ecology and Evolution, University of Chicago, 1101 East 57th Street, Chicago, IL 60637, USA

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BMC Genomics 2013, 14:535  doi:10.1186/1471-2164-14-535

Published: 7 August 2013



As the error rate is high and the distribution of errors across sites is non-uniform in next generation sequencing (NGS) data, it has been a challenge to estimate DNA polymorphism (θ) accurately from NGS data.


By computer simulations, we compare the two methods of data acquisition - sequencing each diploid individual separately and sequencing the pooled sample. Under the current NGS error rate, sequencing each individual separately offers little advantage unless the coverage per individual is high (>20X). We hence propose a new method for estimating θ from pooled samples that have been subjected to two separate rounds of DNA sequencing. Since errors from the two sequencing applications are usually non-overlapping, it is possible to separate low frequency polymorphisms from sequencing errors. Simulation results show that the dual applications method is reliable even when the error rate is high and θ is low.


In studies of natural populations where the sequencing coverage is usually modest (~2X per individual), the dual applications method on pooled samples should be a reasonable choice.

Next generation sequencing; DNA polymorphism; Sequencing error; Pooled sample; Dual sequencing applications