Cytosine Variant Calling with High-throughput Nanopore Sequencing

Abstract

Chemical modifications to DNA regulate cellular state and function. The Oxford Nanopore MinION is a portable single-molecule DNA sequencer that can sequence long fragments of genomic DNA. Here we show that the MinION can be used to detect and map two chemical modifications cytosine, 5-methylcytosine and 5-hydroxymethylcytosine. We present a probabilistic method that enables expansion of the nucleotide alphabet to include bases containing chemical modifications. Our results on synthetic DNA show that individual cytosine base modifications can be classified with accuracy up to 95% in a three-way comparison and 98% in a two-way comparison.\n\nStatement of SignificanceNanopore-based sequencing technology can produce long reads from unamplified genomic DNA, potentially allowing the characterization of chemical modifications and non-canonical DNA nucleotides as they occur in the cell. As the throughput of nanopore sequencers improves, simultaneous detection of multiple epigenetic modifications to cytosines will become an important capability of these devices. Here we present a statistical model that allows the Oxford Nanopore Technologies MinION to be used for detecting chemical modifications to cytosine using standard DNA preparation and sequencing techniques. Our method is based on modeling the ionic current due to DNA k-mers with a variable-order hidden Markov model where the emissions are distributed according to a hierarchical Dirichlet process mixture of normal distributions. This method provides a principled way to expand the nucleotide alphabet to allow for variant calling of modified bases.