Single-Molecule Real-Time (SMRT™) DNA sequencing is unique in that nucleotide incorporation events are monitored in real time, leading to a wealth of kinetic information in addition to the extraction of the primary DNA sequence. The dynamics of the DNA polymerase that is observed adds an additional dimension of sequence-dependent information, and can be used to learn more about the molecule under study. First, the primary sequence itself can be determined more accurately. The kinetic data can be used to corroborate or overturn consensus calls and even enable calling bases in problematic sequence contexts. Second, using the kinetic information we can detect and discriminate nearly every known chemical base modification as a by-product of ordinary sequencing. We will show examples of applying these new capabilities, including a survey of the locations of 5-hydroxymethylcytosine (5-hmC) with single base-pair and DNA strand resolution across mammalian genomes, and an exploration of the mitochondrial genome for known and novel base modifications. We will show our progress towards a generic algorithm for exploiting kinetic information for any of these purposes.
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