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

He Z, Li X, Ling S, Fu YX, Hungate E, Shi S, Wu CI - BMC Genomics (2013)

Bottom Line: 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.Under the current NGS error rate, sequencing each individual separately offers little advantage unless the coverage per individual is high (>20X).Since errors from the two sequencing applications are usually non-overlapping, it is possible to separate low frequency polymorphisms from sequencing errors.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH
θ estimation of dual applications for different region length. The θ value of simulation data is set to 1 per kb. The sequencing error rate is set to 0.005. Singletons are discarded in the estimation (S>1). The length of each error bar is 2 times the standard deviation. The means (and the standard deviations) of θ are estimated from 1000 replicates.
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Figure 3: θ estimation of dual applications for different region length. The θ value of simulation data is set to 1 per kb. The sequencing error rate is set to 0.005. Singletons are discarded in the estimation (S>1). The length of each error bar is 2 times the standard deviation. The means (and the standard deviations) of θ are estimated from 1000 replicates.

Mentions: In Figure 3, we used different region lengths to test the dual applications method. The estimations of θ is acceptable even when the region is small (e.g. 10 kb). For a 40 kb region (the real number of S is about 180), the standard deviation of θ estimates is account for only 5% of the real θ.


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

He Z, Li X, Ling S, Fu YX, Hungate E, Shi S, Wu CI - BMC Genomics (2013)

θ estimation of dual applications for different region length. The θ value of simulation data is set to 1 per kb. The sequencing error rate is set to 0.005. Singletons are discarded in the estimation (S>1). The length of each error bar is 2 times the standard deviation. The means (and the standard deviations) of θ are estimated from 1000 replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750404&req=5

Figure 3: θ estimation of dual applications for different region length. The θ value of simulation data is set to 1 per kb. The sequencing error rate is set to 0.005. Singletons are discarded in the estimation (S>1). The length of each error bar is 2 times the standard deviation. The means (and the standard deviations) of θ are estimated from 1000 replicates.
Mentions: In Figure 3, we used different region lengths to test the dual applications method. The estimations of θ is acceptable even when the region is small (e.g. 10 kb). For a 40 kb region (the real number of S is about 180), the standard deviation of θ estimates is account for only 5% of the real θ.

Bottom Line: 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.Under the current NGS error rate, sequencing each individual separately offers little advantage unless the coverage per individual is high (>20X).Since errors from the two sequencing applications are usually non-overlapping, it is possible to separate low frequency polymorphisms from sequencing errors.

View Article: PubMed Central - HTML - PubMed

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

ABSTRACT

Background: 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.

Results: 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.

Conclusions: 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.

Show MeSH