Modeling kinetic rate variation in third generation DNA sequencing data to detect putative modifications to DNA bases

Abstract

Current generation DNA sequencing instruments are moving closer to seamlessly sequencing genomes of entire populations as a routine part of scientific investigation. However, while significant inroads have been made identifying small nucleotide variation and structural variations in DNA that impact phenotypes of interest, progress has not been as dramatic regarding epigenetic changes and base-level damage to DNA, largely due to technological limitations in assaying all known and unknown types of modifications at genome scale. Recently, single-molecule real time (SMRT) sequencing has been reported to identify kinetic variation (KV) events that have been demonstrated to reflect epigenetic changes of every known type, providing a path forward for detecting base modifications as a routine part of sequencing. However, to date no statistical framework has been proposed to enhance the power to detect these events while also controlling for false-positive events. By modeling enzyme kinetics in the neighborhood of an arbitrary location in a genomic region of interest as a conditional random field, we provide a statistical framework for incorporating kinetic information at a test position of interest as well as at neighboring sites that help enhance the power to detect KV events. The performance of this and related models is explored, with the best-performing model applied to plasmid DNA isolated from Escherichia coli and mitochondrial DNA isolated from human brain tissue. We highlight widespread kinetic variation events, some of which strongly associate with known modification events, while others represent putative chemically modified sites of unknown types. [Supplemental material is available for this article.] DNA sequencing carried out using current first and second (or next) generation sequencing (NGS) instruments has achieved significant success in providing DNA sequences in which the A's, G's, C's, and T's comprising any given DNA template of interest are very consistently called at accuracies that exceed 99%, enabling an explosion of whole-genome sequencing applications that promise to transform our understanding of living systems and usher in the era of personalized genomics (Shendure and Ji 2008; Wheeler et al.

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing

“…In SMRT sequencing, the time required for the incorporation of each nucleotide can be monitored, in addition to the specific base selected. Kinetic variation (KV), or variation in the rate at which DNA polymerase incorporates bases into DNA during synthesis, is highly correlated with the presence of modifications in the template 23,24 . Furthermore, different modifications, such as m6A and m5C, are linked to distinct kinetic profiles 22–24 .…”

“…Kinetic variation (KV), or variation in the rate at which DNA polymerase incorporates bases into DNA during synthesis, is highly correlated with the presence of modifications in the template 23,24 . Furthermore, different modifications, such as m6A and m5C, are linked to distinct kinetic profiles 22–24 . Therefore, SMRT sequencing offers the possibility of directly detecting chemical modification (or damage) to nucleotides and discriminating between such changes 22,23 .…”

DNA N6-methyladenine: a new epigenetic mark in eukaryotes?

“…SMRT sequencing provides both accurate sequence reads and determines the kinetics of nucleotide incorporations during synthesis. Different DNA modifications induce unique kinetic signatures that can be used for accurate and sequence-specific detection of modifications 35, 36 . SMRT sequencing has been used to map 6mA and 5mC simultaneously in Escherichia coli 37 and 6mA in C. elegans 15 .…”

Single-Cell Sequencing Technologies: Current and Future

“…Third-generation sequencing technologies enable improved sequencing length (Flusberg et al, 2010) that can facilitate the profiling of long repetitive sequences, alternative splicing, and gene fusion. Direct mapping of 5mC and 5hmC has been achieved using Nanopore-sequencing (Laszlo et al, 2013) and SMRT sequencing technologies (Flusberg et al, 2010;Schadt et al, 2013). Applying third-generation sequencing technology, Ozsolak et al (2009) performed direct RNA sequencing without prior conversion of RNA to cDNA.…”