Abstract
DNA modifications such as methylation and DNA damage can play critical regulatory roles in biological systems. Single molecule, real time (SMRT) sequencing technology generates DNA sequences as well as DNA polymerase kinetic information that can be used for the direct detection of DNA modifications. We demonstrate that local sequence context has a strong impact on DNA polymerase kinetics in the neighborhood of the incorporation site during the DNA synthesis reaction, allowing for the possibility of estimating the expected kinetic rate of the enzyme at the incorporation site using kinetic rate information collected from existing SMRT sequencing data (historical data) covering the same local sequence contexts of interest. We develop an Empirical Bayesian hierarchical model for incorporating historical data. Our results show that the model could greatly increase DNA modification detection accuracy, and reduce requirement of control data coverage. For some DNA modifications that have a strong signal, a control sample is not even needed by using historical data as alternative to control. Thus, sequencing costs can be greatly reduced by using the model. We implemented the model in a R package named seqPatch, which is available at https://github.com/zhixingfeng/seqPatch.
Highlights
Modifications to individual bases like 5-methylcytosine, 5hydroxymethylcytosine, and N6-methyladenine in DNA sequences are an important epigenetic component to the regulation of living systems, from individual genes to cellular function
The kinetic information is sensitive to DNA modifications in the sequenced DNA template, and can be used for detecting a wide range of DNA modification types
We proposed a hierarchical model, which can incorporate existing SMRT sequencing data to increase detection accuracy and reduce coverage requirement of control sample or even avoid the need of a control sample in some cases
Summary
Modifications to individual bases like 5-methylcytosine, 5hydroxymethylcytosine, and N6-methyladenine in DNA sequences are an important epigenetic component to the regulation of living systems, from individual genes to cellular function. In SMRT sequencing, each base identity is read when fluorescently labeled nucleotides are incorporated into a DNA sequence being synthesized by DNA polymerase [4]. In this case, because the incorporation events are being directly observed in real time, the duration between the pulses of light (referred to as inter-pulse duration or IPD) that indicate an incorporation event can be precisely measured. IPD measures are a direct reflection of the DNA polymerase kinetics This kinetic parameter for the enzyme has been shown to be sensitive to a wide range of DNA modification events, including 5-methylcytosine, 5-hydroxymethylcytosine, and N6-methyladenocine [1,2,3], where variations in the kinetics are predictive of modification events
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